Lipids and lipid nanoparticle formulations for delivery of nucleic acids

ABSTRACT

Compounds are provided having the following structure (I) or (II): or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R, R 1 , R 2 , L, X, L, G 1 , G 2 , Z, a 1 , a 2  and n are independently as defined herein for each of structures (I) and (II). Use of the compounds as a component of lipid nanoparticle formulations for delivery of a therapeutic agent, compositions comprising the compounds and methods for their use and preparation are also provided.

BACKGROUND Technical Field

The present invention generally relates to novel cationic lipids thatcan be used in combination with other lipid components, such as neutrallipids, cholesterol and polymer conjugated lipids, to form lipidnanoparticles with oligonucleotides, to facilitate the intracellulardelivery of therapeutic nucleic acids (e.g. oligonucleotides, messengerRNA) both in vitro and in vivo.

Description of the Related Art

There are many challenges associated with the delivery of nucleic acidsto affect a desired response in a biological system. Nucleic acid basedtherapeutics have enormous potential but there remains a need for moreeffective delivery of nucleic acids to appropriate sites within a cellor organism in order to realize this potential. Therapeutic nucleicacids include, e.g., messenger RNA (mRNA), antisense oligonucleotides,ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids,antagomir, antimir, mimic, supermir, and aptamers. Some nucleic acids,such as mRNA or plasmids, can be used to effect expression of specificcellular products as would be useful in the treatment of, for example,diseases related to a deficiency of a protein or enzyme. The therapeuticapplications of translatable nucleotide delivery are extremely broad asconstructs can be synthesized to produce any chosen protein sequence,whether or not indigenous to the system. The expression products of thenucleic acid can augment existing levels of protein, replace missing ornon-functional versions of a protein, or introduce new protein andassociated functionality in a cell or organism.

Some nucleic acids, such as miRNA inhibitors, can be used to effectexpression of specific cellular products that are regulated by miRNA aswould be useful in the treatment of, for example, diseases related todeficiency of protein or enzyme. The therapeutic applications of miRNAinhibition are extremely broad as constructs can be synthesized toinhibit one or more miRNA that would in turn regulate the expression ofmRNA products. The inhibition of endogenous miRNA can augment itsdownstream target endogenous protein expression and restore properfunction in a cell or organism as a means to treat disease associated toa specific miRNA or a group of miRNA.

Other nucleic acids can down-regulate intracellular levels of specificmRNA and, as a result, down-regulate the synthesis of the correspondingproteins through processes such as RNA interference (RNAi) orcomplementary binding of antisense RNA. The therapeutic applications ofantisense oligonucleotide and RNAi are also extremely broad, sinceoligonucleotide constructs can be synthesized with any nucleotidesequence directed against a target mRNA. Targets may include mRNAs fromnormal cells, mRNAs associated with disease-states, such as cancer, andmRNAs of infectious agents, such as viruses. To date, antisenseoligonucleotide constructs have shown the ability to specificallydown-regulate target proteins through degradation of the cognate mRNA inboth in vitro and in vivo models. In addition, antisense oligonucleotideconstructs are currently being evaluated in clinical studies.

However, two problems currently face the use of oligonucleotides intherapeutic contexts. First, free RNAs are susceptible to nucleasedigestion in plasma. Second, free RNAs have limited ability to gainaccess to the intracellular compartment where the relevant translationmachinery resides. Lipid nanoparticles formed from cationic lipids withother lipid components, such as neutral lipids, cholesterol, PEG,PEGylated lipids, and oligonucleotides have been used to blockdegradation of the RNAs in plasma and facilitate the cellular uptake ofthe oligonucleotides.

There remains a need for improved cationic lipids and lipidnanoparticles for the delivery of oligonucleotides. Preferably, theselipid nanoparticles would provide optimal drug:lipid ratios, protect thenucleic acid from degradation and clearance in serum, be suitable forsystemic or local delivery, and provide intracellular delivery of thenucleic acid. In addition, these lipid-nucleic acid particles should bewell-tolerated and provide an adequate therapeutic index, such thatpatient treatment at an effective dose of the nucleic acid is notassociated with unacceptable toxicity and/or risk to the patient. Thepresent invention provides these and related advantages.

BRIEF SUMMARY

In brief, the present invention provides lipid compounds, includingstereoisomers, pharmaceutically acceptable salts or tautomers thereof,which can be used alone or in combination with other lipid componentssuch as neutral lipids, charged lipids, steroids (including for example,all sterols) and/or their analogs, and/or polymer conjugated lipids toform lipid nanoparticles for the delivery of therapeutic agents. In someinstances, the lipid nanoparticles are used to deliver nucleic acidssuch as antisense and/or messenger RNA. Methods for use of such lipidnanoparticles for treatment of various diseases or conditions, such asthose caused by infectious entities and/or insufficiency of a protein,are also provided.

In one embodiment, compounds having the following structure (I) or (II)are provided:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein R, R¹, R², L, X, L, G², Z, a¹, a² and n are independently asdefined herein for each of structures (I) and (II).

Pharmaceutical compositions comprising one or more of the foregoingcompounds of structure (I) or (II) and a therapeutic agent are alsoprovided. In some embodiments, the pharmaceutical compositions furthercomprise one or more components selected from neutral lipids, chargedlipids, steroids and polymer conjugated lipids. Such compositions areuseful for formation of lipid nanoparticles for the delivery of thetherapeutic agent.

In other embodiments, the present invention provides a method foradministering a therapeutic agent to a patient in need thereof, themethod comprising preparing a composition of lipid nanoparticlescomprising the compound of structure (I) or (II) and a therapeutic agentand delivering the composition to the patient.

These and other aspects of the invention will be apparent upon referenceto the following detailed description.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

In the figures, identical reference numbers identify similar elements.The sizes and relative positions of elements in the figures are notnecessarily drawn to scale and some of these elements are arbitrarilyenlarged and positioned to improve figure legibility. Further, theparticular shapes of the elements as drawn are not intended to conveyany information regarding the actual shape of the particular elements,and have been solely selected for ease of recognition in the figures.

FIG. 1 shows time course of luciferase expression in mouse liver.

FIG. 2 illustrates the calculation of pK_(a) for MC3 as a representativeexample relevant to the disclosed lipids.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of theinvention. However, one skilled in the art will understand that theinvention may be practiced without these details.

The present invention is based, in part, upon the discovery of novelcationic (amino) lipids that provide advantages when used in lipidnanoparticles for the in vivo delivery of an active or therapeutic agentsuch as a nucleic acid into a cell of a mammal. In particular,embodiments of the present invention provide nucleic acid-lipidnanoparticle compositions comprising one or more of the novel cationiclipids described herein that provide increased activity of the nucleicacid and improved tolerability of the compositions in vivo, resulting ina significant increase in the therapeutic index as compared to nucleicacid-lipid nanoparticle compositions previously described.

In particular embodiments, the present invention provides novel cationiclipids that enable the formulation of improved compositions for the invitro and in vivo delivery of mRNA and/or other oligonucleotides. Insome embodiments, these improved lipid nanoparticle compositions areuseful for expression of protein encoded by mRNA. In other embodiments,these improved lipid nanoparticles compositions are useful forupregulation of endogenous protein expression by delivering miRNAinhibitors targeting one specific miRNA or a group of miRNA regulatingone target mRNA or several mRNA. In other embodiments, these improvedlipid nanoparticle compositions are useful for down-regulating (e.g.,silencing) the protein levels and/or mRNA levels of target genes. Insome other embodiments, the lipid nanoparticles are also useful fordelivery of mRNA and plasmids for expression of transgenes. In yet otherembodiments, the lipid nanoparticle compositions are useful for inducinga pharmacological effect resulting from expression of a protein, e.g.,increased production of red blood cells through the delivery of asuitable erythropoietin mRNA, or protection against infection throughdelivery of mRNA encoding for a suitable antigen or antibody.

The lipid nanoparticles and compositions of the present invention may beused for a variety of purposes, including the delivery of encapsulatedor associated (e.g., complexed) therapeutic agents such as nucleic acidsto cells, both in vitro and in vivo. Accordingly, embodiments of thepresent invention provide methods of treating or preventing diseases ordisorders in a subject in need thereof by contacting the subject with alipid nanoparticle that encapsulates or is associated with a suitabletherapeutic agent, wherein the lipid nanoparticle comprises one or moreof the novel cationic lipids described herein.

As described herein, embodiments of the lipid nanoparticles of thepresent invention are particularly useful for the delivery of nucleicacids, including, e.g., mRNA, antisense oligonucleotide, plasmid DNA,microRNA (miRNA), miRNA inhibitors (antagomirs/antimirs),messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalentRNA, dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, thelipid nanoparticles and compositions of the present invention may beused to induce expression of a desired protein both in vitro and in vivoby contacting cells with a lipid nanoparticle comprising one or morenovel cationic lipids described herein, wherein the lipid nanoparticleencapsulates or is associated with a nucleic acid that is expressed toproduce the desired protein (e.g., a messenger RNA or plasmid encodingthe desired protein) or inhibit processes that terminate expression ofmRNA (e.g., miRNA inhibitors). Alternatively, the lipid nanoparticlesand compositions of the present invention may be used to decrease theexpression of target genes and proteins both in vitro and in vivo bycontacting cells with a lipid nanoparticle comprising one or more novelcationic lipids described herein, wherein the lipid nanoparticleencapsulates or is associated with a nucleic acid that reduces targetgene expression (e.g., an antisense oligonucleotide or small interferingRNA (siRNA)). The lipid nanoparticles and compositions of the presentinvention may also be used for co-delivery of different nucleic acids(e.g. mRNA and plasmid DNA) separately or in combination, such as may beuseful to provide an effect requiring colocalization of differentnucleic acids (e.g. mRNA encoding for a suitable gene modifying enzymeand DNA segment(s) for incorporation into the host genome).

Nucleic acids for use with this invention may be prepared according toany available technique. For mRNA, the primary methodology ofpreparation is, but not limited to, enzymatic synthesis (also termed invitro transcription) which currently represents the most efficientmethod to produce long sequence-specific mRNA. In vitro transcriptiondescribes a process of template-directed synthesis of RNA molecules froman engineered DNA template comprised of an upstream bacteriophagepromoter sequence (e.g. including but not limited to that from the T7,T3 and SP6 coliphage) linked to a downstream sequence encoding the geneof interest. Template DNA can be prepared for in vitro transcriptionfrom a number of sources with appropriate techniques which are wellknown in the art including, but not limited to, plasmid DNA andpolymerase chain reaction amplification (see Linpinsel, J. L. and Conn,G. L., General protocols for preparation of plasmid DNA template andBowman, J. C., Azizi, B., Lenz, T. K., Ray, P., and Williams, L. D. inRNA in vitro transcription and RNA purification by denaturing PAGE inRecombinant and in vitro RNA syntheses Methods v. 941 Conn G. L. (ed),New York, N.Y. Humana Press, 2012)

Transcription of the RNA occurs in vitro using the linearized DNAtemplate in the presence of the corresponding RNA polymerase andadenosine, guanosine, uridine and cytidine ribonucleoside triphosphates(rNTPs) under conditions that support polymerase activity whileminimizing potential degradation of the resultant mRNA transcripts. Invitro transcription can be performed using a variety of commerciallyavailable kits including, but not limited to RiboMax Large Scale RNAProduction System (Promega), MegaScript Transcription kits (LifeTechnologies) as well as with commercially available reagents includingRNA polymerases and rNTPs. The methodology for in vitro transcription ofmRNA is well known in the art. (see, e.g. Losick, R., 1972, In vitrotranscription, Ann Rev Biochem v.41 409-46; Kamakaka, R. T. and Kraus,W. L. 2001. In Vitro Transcription. Current Protocols in Cell Biology.2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B.,(2010) Synthesis ofRNA by In Vitro Transcription in RNA in Methods in Molecular Biology v.703 (Neilson, H. Ed), New York, N.Y. Humana Press, 2010; Brunelle, J. L.and Green, R., 2013, Chapter Five—In vitro transcription from plasmid orPCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of whichare incorporated herein by reference).

The desired in vitro transcribed mRNA is then purified from theundesired components of the transcription or associated reactions(including unincorporated rNTPs, protein enzyme, salts, short RNAoligos, etc.). Techniques for the isolation of the mRNA transcripts arewell known in the art. Well known procedures include phenol/chloroformextraction or precipitation with either alcohol (ethanol, isopropanol)in the presence of monovalent cations or lithium chloride. Additional,non-limiting examples of purification procedures which can be usedinclude size exclusion chromatography (Lukaysky, P. J. and Puglisi, J.D., 2004, Large-scale preparation and purification ofpolyacrylamide-free RNA oligonucleotides, RNA v.10, 889-893),silica-based affinity chromatography and polyacrylamide gelelectrophoresis (Bowman, J. C., Azizi, B., Lenz, T. K., Ray, P., andWilliams, L. D. in RNA in vitro transcription and RNA purification bydenaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941Conn G. L. (ed), New York, N.Y. Humana Press, 2012). Purification can beperformed using a variety of commercially available kits including, butnot limited to SV Total Isolation System (Promega) and In VitroTranscription Cleanup and Concentration Kit (Norgen Biotek).

Furthermore, while reverse transcription can yield large quantities ofmRNA, the products can contain a number of aberrant RNA impuritiesassociated with undesired polymerase activity which may need to beremoved from the full-length mRNA preparation. These include short RNAsthat result from abortive transcription initiation as well asdouble-stranded RNA (dsRNA) generated by RNA-dependent RNA polymeraseactivity, RNA-primed transcription from RNA templates andself-complementary 3′ extension. It has been demonstrated that thesecontaminants with dsRNA structures can lead to undesiredimmunostimulatory activity through interaction with various innateimmune sensors in eukaryotic cells that function to recognize specificnucleic acid structures and induce potent immune responses. This inturn, can dramatically reduce mRNA translation since protein synthesisis reduced during the innate cellular immune response. Therefore,additional techniques to remove these dsRNA contaminants have beendeveloped and are known in the art including but not limited toscaleable HPLC purification (see e.g. Kariko, K., Muramatsu, H., Ludwig,J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLCpurification eliminates immune activation and improves translation ofnucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 e142;Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLCPurification of in vitro transcribed long RNA in Synthetic Messenger RNAand Cell Metabolism Modulation in Methods in Molecular Biology v.969(Rabinovich, P.H. Ed), 2013). HPLC purified mRNA has been reported to betranslated at much greater levels, particularly in primary cells and invivo.

A significant variety of modifications have been described in the artwhich are used to alter specific properties of in vitro transcribedmRNA, and improve its utility. These include, but are not limited tomodifications to the 5′ and 3′ termini of the mRNA. Endogenouseukaryotic mRNA typically contain a cap structure on the 5′-end of amature molecule which plays an important role in mediating binding ofthe mRNA Cap Binding Protein (CBP), which is in turn responsible forenhancing mRNA stability in the cell and efficiency of mRNA translation.Therefore, highest levels of protein expression are achieved with cappedmRNA transcripts. The 5′-cap contains a 5′-5′-triphosphate linkagebetween the 5′-most nucleotide and guanine nucleotide. The conjugatedguanine nucleotide is methylated at the N7 position. Additionalmodifications include methylation of the ultimate and penultimate most5′-nucleotides on the 2′-hydroxyl group.

Multiple distinct cap structures can be used to generate the 5′-cap ofin vitro transcribed synthetic mRNA. 5′-capping of synthetic mRNA can beperformed co-transcriptionally with chemical cap analogs (i.e. cappingduring in vitro transcription). For example, the Anti-Reverse Cap Analog(ARCA) cap contains a 5′-5′-triphosphate guanine-guanine linkage whereone guanine contains an N7 methyl group as well as a 3′-O-methyl group.However, up to 20% of transcripts remain uncapped during thisco-transcriptional process and the synthetic cap analog is not identicalto the 5′-cap structure of an authentic cellular mRNA, potentiallyreducing translatability and cellular stability. Alternatively,synthetic mRNA molecules may also be enzymatically cappedpost-transcriptionally. These may generate a more authentic 5′-capstructure that more closely mimics, either structurally or functionally,the endogenous 5′-cap which have enhanced binding of cap bindingproteins, increased half-life, reduced susceptibility to 5′endonucleases and/or reduced 5′ decapping. Numerous synthetic 5′-capanalogs have been developed and are known in the art to enhance mRNAstability and translatability (see e.g. Grudzien-Nogalska, E., Kowalska,J., Su, W., Kuhn, A. N., Slepenkov, S. V., Darynkiewicz, E., Sahin, U.,Jemielity, J., and Rhoads, R. E., Synthetic mRNAs with superiortranslation and stability properties in Synthetic Messenger RNA and CellMetabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich,P.H. Ed), 2013).

On the 3′-terminus, a long chain of adenine nucleotides (poly-A tail) isnormally added to mRNA molecules during RNA processing. Immediatelyafter transcription, the 3′ end of the transcript is cleaved to free a3′ hydroxyl to which poly-A polymerase adds a chain of adeninenucleotides to the RNA in a process called polyadenylation. The poly-Atail has been extensively shown to enhance both translational efficiencyand stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A),poly (A) binding protein and the regulation of mRNA stability, TrendsBio Sci v. 14 373-377; Guhaniyogi, J. And Brewer, G., 2001, Regulationof mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M.And Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard ineukaryotes, scavenger in bacteria, Cell, v.111, 611-613).

Poly (A) tailing of in vitro transcribed mRNA can be achieved usingvarious approaches including, but not limited to, cloning of a poly (T)tract into the DNA template or by post-transcriptional addition usingPoly (A) polymerase. The first case allows in vitro transcription ofmRNA with poly (A) tails of defined length, depending on the size of thepoly (T) tract, but requires additional manipulation of the template.The latter case involves the enzymatic addition of a poly (A) tail to invitro transcribed mRNA using poly (A) polymerase which catalyzes theincorporation of adenine residues onto the 3′termini of RNA, requiringno additional manipulation of the DNA template, but results in mRNA withpoly(A) tails of heterogeneous length. 5′-capping and 3′-poly (A)tailing can be performed using a variety of commercially available kitsincluding, but not limited to Poly (A) Polymerase Tailing kit(EpiCenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit(Life Technologies) as well as with commercially available reagents,various ARCA caps, Poly (A) polymerase, etc.

In addition to 5′ cap and 3′ poly adenylation, other modifications ofthe in vitro transcripts have been reported to provide benefits asrelated to efficiency of translation and stability. It is well known inthe art that pathogenic DNA and RNA can be recognized by a variety ofsensors within eukaryotes and trigger potent innate immune responses.The ability to discriminate between pathogenic and self DNA and RNA hasbeen shown to be based, at least in part, on structure and nucleosidemodifications since most nucleic acids from natural sources containmodified nucleosides. In contrast, in vitro synthesized RNA lacks thesemodifications, thus rendering it immunostimulatory which in turn caninhibit effective mRNA translation as outlined above. The introductionof modified nucleosides into in vitro transcribed mRNA can be used toprevent recognition and activation of RNA sensors, thus mitigating thisundesired immunostimulatory activity and enhancing translation capacity(see e.g. Kariko, K. And Weissman, D. 2007, Naturally occurringnucleoside modifications suppress the immunostimulatory activity of RNA:implication for therapeutic RNA development, Curr Opin Drug DiscovDevel, v.10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K.,In vitro transcription of long RNA containing modified nucleosides inSynthetic Messenger RNA and Cell Metabolism Modulation in Methods inMolecular Biology v.969 (Rabinovich, P.H. Ed), 2013); Kariko, K.,Muramatsu, H., Welsh, F. A., Ludwig, J., Kato, H., Akira, S., Weissman,D., 2008, Incorporation of Pseudouridine Into mRNA Yields SuperiorNonimmunogenic Vector With Increased Translational Capacity andBiological Stability, Mol Ther v.16, 1833-1840. The modified nucleosidesand nucleotides used in the synthesis of modified RNAs can be preparedmonitored and utilized using general methods and procedures known in theart. A large variety of nucleoside modifications are available that maybe incorporated alone or in combination with other modified nucleosidesto some extent into the in vitro transcribed mRNA (see e.g.US2012/0251618). In vitro synthesis of nucleoside-modified mRNA has beenreported to have reduced ability to activate immune sensors with aconcomitant enhanced translational capacity.

Other components of mRNA which can be modified to provide benefit interms of translatability and stability include the 5′ and 3′untranslated regions (UTR). Optimization of the UTRs (favorable 5′ and3′ UTRs can be obtained from cellular or viral RNAs), either both orindependently, have been shown to increase mRNA stability andtranslational efficiency of in vitro transcribed mRNA (see e.g. Pardi,N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription oflong RNA containing modified nucleosides in Synthetic Messenger RNA andCell Metabolism Modulation in Methods in Molecular Biology v.969(Rabinovich, P.H. Ed), 2013).

In addition to mRNA, other nucleic acid payloads may be used for thisinvention. For oligonucleotides, methods of preparation include but arenot limited to chemical synthesis and enzymatic, chemical cleavage of alonger precursor, in vitro transcription as described above, etc.Methods of synthesizing DNA and RNA nucleotides are widely used and wellknown in the art (see, e.g. Gait, M. J. (ed.) Oligonucleotide synthesis:a practical approach, Oxford [Oxfordshire], Washington, D.C.: IRL Press,1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods andapplications, Methods in Molecular Biology, v. 288 (Clifton, N.J.)Totowa, N.J.: Humana Press, 2005; both of which are incorporated hereinby reference).

For plasmid DNA, preparation for use with this invention commonlyutilizes but is not limited to expansion and isolation of the plasmidDNA in vitro in a liquid culture of bacteria containing the plasmid ofinterest. The presence of a gene in the plasmid of interest that encodesresistance to a particular antibiotic (penicillin, kanamycin, etc.)allows those bacteria containing the plasmid of interest to selectivelygrow in antibiotic-containing cultures. Methods of isolating plasmid DNAare widely used and well known in the art (see, e.g. Heilig, J., Elbing,K. L. and Brent, R (2001) Large-Scale Preparation of Plasmid DNA.Current Protocols in Molecular Biology. 41:11:1.7:1.7.1-1.7.16; Rozkov,A., Larsson, B., Gillstrom, S., Bjornestedt, R. and Schmidt, S. R.(2008), Large-scale production of endotoxin-free plasmids for transientexpression in mammalian cell culture. Biotechnol. Bioeng., 99: 557-566;and U.S. Pat. No. 6,197,553B1). Plasmid isolation can be performed usinga variety of commercially available kits including, but not limited toPlasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo) and PureYieldMaxiPrep (Promega) kits as well as with commercially available reagents.

Various exemplary embodiments of the cationic lipids of the presentinvention, lipid nanoparticles and compositions comprising the same, andtheir use to deliver active (e.g. therapeutic agents), such as nucleicacids, to modulate gene and protein expression, are described in furtherdetail below.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open andinclusive sense, that is, as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. As used in the specification andclaims, the singular form “a”, “an” and “the” include plural referencesunless the context clearly dictates otherwise.

The phrase “induce expression of a desired protein” refers to theability of a nucleic acid to increase expression of the desired protein.To examine the extent of protein expression, a test sample (e.g. asample of cells in culture expressing the desired protein) or a testmammal (e.g. a mammal such as a human or an animal model such as arodent (e.g. mouse) or a non-human primate (e.g., monkey) model) iscontacted with a nucleic acid (e.g. nucleic acid in combination with alipid of the present invention). Expression of the desired protein inthe test sample or test animal is compared to expression of the desiredprotein in a control sample (e.g. a sample of cells in cultureexpressing the desired protein) or a control mammal (e.g., a mammal suchas a human or an animal model such as a rodent (e.g. mouse) or non-humanprimate (e.g. monkey) model) that is not contacted with or administeredthe nucleic acid. When the desired protein is present in a controlsample or a control mammal, the expression of a desired protein in acontrol sample or a control mammal may be assigned a value of 1.0. Inparticular embodiments, inducing expression of a desired protein isachieved when the ratio of desired protein expression in the test sampleor the test mammal to the level of desired protein expression in thecontrol sample or the control mammal is greater than 1, for example,about 1.1, 1.5, 2.0. 5.0 or 10.0. When a desired protein is not presentin a control sample or a control mammal, inducing expression of adesired protein is achieved when any measurable level of the desiredprotein in the test sample or the test mammal is detected. One ofordinary skill in the art will understand appropriate assays todetermine the level of protein expression in a sample, for example dotblots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, and phenotypic assays, or assaysbased on reporter proteins that can produce fluorescence or luminescenceunder appropriate conditions.

The phrase “inhibiting expression of a target gene” refers to theability of a nucleic acid to silence, reduce, or inhibit the expressionof a target gene. To examine the extent of gene silencing, a test sample(e.g. a sample of cells in culture expressing the target gene) or a testmammal (e.g. a mammal such as a human or an animal model such as arodent (e.g. mouse) or a non-human primate (e.g. monkey) model) iscontacted with a nucleic acid that silences, reduces, or inhibitsexpression of the target gene. Expression of the target gene in the testsample or test animal is compared to expression of the target gene in acontrol sample (e.g. a sample of cells in culture expressing the targetgene) or a control mammal (e.g. a mammal such as a human or an animalmodel such as a rodent (e.g. mouse) or non-human primate (e.g. monkey)model) that is not contacted with or administered the nucleic acid. Theexpression of the target gene in a control sample or a control mammalmay be assigned a value of 100%. In particular embodiments, silencing,inhibition, or reduction of expression of a target gene is achieved whenthe level of target gene expression in the test sample or the testmammal relative to the level of target gene expression in the controlsample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Inother words, the nucleic acids are capable of silencing, reducing, orinhibiting the expression of a target gene by at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% in a test sample or a test mammal relative to thelevel of target gene expression in a control sample or a control mammalnot contacted with or administered the nucleic acid. Suitable assays fordetermining the level of target gene expression include, withoutlimitation, examination of protein or mRNA levels using techniques knownto those of skill in the art, such as, e.g., dot blots, northern blots,in situ hybridization, ELISA, immunoprecipitation, enzyme function, aswell as phenotypic assays known to those of skill in the art.

An “effective amount” or “therapeutically effective amount” of an activeagent or therapeutic agent such as a therapeutic nucleic acid is anamount sufficient to produce the desired effect, e.g. an increase orinhibition of expression of a target sequence in comparison to thenormal expression level detected in the absence of the nucleic acid. Anincrease in expression of a target sequence is achieved when anymeasurable level is detected in the case of an expression product thatis not present in the absence of the nucleic acid. In the case where theexpression product is present at some level prior to contact with thenucleic acid, an in increase in expression is achieved when the foldincrease in value obtained with a nucleic acid such as mRNA relative tocontrol is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000,5000, 10000 or greater. Inhibition of expression of a target gene ortarget sequence is achieved when the value obtained with a nucleic acidsuch as antisense oligonucleotide relative to the control is about 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of atarget gene or target sequence include, e.g., examination of protein orRNA levels using techniques known to those of skill in the art such asdot blots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, fluorescence or luminescence ofsuitable reporter proteins, as well as phenotypic assays known to thoseof skill in the art.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA, RNA, and hybrids thereof. DNA maybe in the form of antisense molecules, plasmid DNA, cDNA, PCR products,or vectors. RNA may be in the form of small hairpin RNA (shRNA),messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA,dicer substrate RNA or viral RNA (vRNA), and combinations thereof.Nucleic acids include nucleic acids containing known nucleotide analogsor modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, and which have similarbinding properties as the reference nucleic acid. Examples of suchanalogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), alleles, orthologs, single nucleotide polymorphisms, andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka etal., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell.Probes, 8:91-98 (1994)). “Nucleotides” contain a sugar deoxyribose (DNA)or ribose (RNA), a base, and a phosphate group. Nucleotides are linkedtogether through the phosphate groups. “Bases” include purines andpyrimidines, which further include natural compounds adenine, thymine,guanine, cytosine, uracil, inosine, and natural analogs, and syntheticderivatives of purines and pyrimidines, which include, but are notlimited to, modifications which place new reactive groups such as, butnot limited to, amines, alcohols, thiols, carboxylates, andalkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are generallycharacterized by being poorly soluble in water, but soluble in manyorganic solvents. They are usually divided into at least three classes:(1) “simple lipids,” which include fats and oils as well as waxes; (2)“compound lipids,” which include phospholipids and glycolipids; and (3)“derived lipids” such as steroids.

A “steroid” is a compound comprising the following carbon skeleton:

Non-limiting examples of steroids include cholesterol, and the like.

A “cationic lipid” refers to a lipid capable of being positivelycharged. Exemplary cationic lipids include one or more amine group(s)which bears the positive charge. Preferred cationic lipids are ionizablesuch that they can exist in a positively charged or neutral formdepending on pH. The ionization of the cationic lipid affects thesurface charge of the lipid nanoparticle under different pH conditions.This charge state can influence plasma protein absorption, bloodclearance and tissue distribution (Semple, S. C., et al., Adv. DrugDeliv Rev 32:3-17 (1998)) as well as the ability to form endosomolyticnon-bilayer structures (Hafez, I. M., et al., Gene Ther 8:1188-1196(2001)) critical to the intracellular delivery of nucleic acids.

The term “polymer conjugated lipid” refers to a molecule comprising botha lipid portion and a polymer portion. An example of a polymerconjugated lipid is a pegylated lipid. The term “pegylated lipid” refersto a molecule comprising both a lipid portion and a polyethylene glycolportion. Pegylated lipids are known in the art and include1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) andthe like.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, but are not limited to,phosphotidylcholines such as 1,2-Distearoyl-sn-glycero-3-phosphocholine(DSPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),phophatidylethanolamines such as1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins(SM), ceramides, steroids such as sterols and their derivatives. Neutrallipids may be synthetic or naturally derived.

The term “charged lipid” refers to any of a number of lipid species thatexist in either a positively charged or negatively charged formindependent of the pH within a useful physiological range e.g. pH ˜3 topH ˜9. Charged lipids may be synthetic or naturally derived. Examples ofcharged lipids include phosphatidylserines, phosphatidic acids,phosphatidylglycerols, phosphatidylinositols, sterol hemisuccinates,dialkyl trimethylammonium-propanes, (e.g. DOTAP, DOTMA), dialkyldimethylaminopropanes, ethyl phosphocholines, dimethylaminoethanecarbamoyl sterols (e.g. DC-Chol).

The term “lipid nanoparticle” refers to particles having at least onedimension on the order of nanometers (e.g., 1-1,000 nm) which includeone or more of the compounds of structure (I) or (II) or other specifiedcationic lipids. In some embodiments, lipid nanoparticles are includedin a formulation that can be used to deliver an active agent ortherapeutic agent, such as a nucleic acid (e.g., mRNA) to a target siteof interest (e.g., cell, tissue, organ, tumor, and the like). In someembodiments, the lipid nanoparticles of the invention comprise a nucleicacid. Such lipid nanoparticles typically comprise a compound ofstructure (I) or (II) and one or more excipient selected from neutrallipids, charged lipids, steroids and polymer conjugated lipids. In someembodiments, the active agent or therapeutic agent, such as a nucleicacid, may be encapsulated in the lipid portion of the lipid nanoparticleor an aqueous space enveloped by some or all of the lipid portion of thelipid nanoparticle, thereby protecting it from enzymatic degradation orother undesirable effects induced by the mechanisms of the host organismor cells e.g. an adverse immune response.

In various embodiments, the lipid nanoparticles have a mean diameter offrom about 30 nm to about 150 nm, from about 40 nm to about 150 nm, fromabout 50 nm to about 150 nm, from about 60 nm to about 130 nm, fromabout 70 nm to about 110 nm, from about 70 nm to about 100 nm, fromabout 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm,and are substantially non-toxic. In certain embodiments, nucleic acids,when present in the lipid nanoparticles, are resistant in aqueoussolution to degradation with a nuclease. Lipid nanoparticles comprisingnucleic acids and their method of preparation are disclosed in, e.g.,U.S. Patent Publication Nos. 2004/0142025, 2007/0042031 and PCT Pub.Nos. WO 2013/016058 and WO 2013/086373, the full disclosures of whichare herein incorporated by reference in their entirety for all purposes.

As used herein, “lipid encapsulated” refers to a lipid nanoparticle thatprovides an active agent or therapeutic agent, such as a nucleic acid(e.g., mRNA), with full encapsulation, partial encapsulation, or both.In an embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated inthe lipid nanoparticle.

As used herein, the term “aqueous solution” refers to a compositioncomprising water.

“Serum-stable” in relation to nucleic acid-lipid nanoparticles meansthat the nucleotide is not significantly degraded after exposure to aserum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of a therapeuticproduct that can result in a broad exposure of an active agent within anorganism. Some techniques of administration can lead to the systemicdelivery of certain agents, but not others. Systemic delivery means thata useful, preferably therapeutic, amount of an agent is exposed to mostparts of the body. Systemic delivery of lipid nanoparticles can be byany means known in the art including, for example, intravenous,intra-arterial, subcutaneous, and intraperitoneal delivery. In someembodiments, systemic delivery of lipid nanoparticles is by intravenousdelivery.

“Local delivery,” as used herein, refers to delivery of an active agentdirectly to a target site within an organism. For example, an agent canbe locally delivered by direct injection into a disease site such as atumor, other target site such as a site of inflammation, or a targetorgan such as the liver, heart, pancreas, kidney, and the like. Localdelivery can also include topical applications or localized injectiontechniques such as intramuscular, subcutaneous or intradermal injection.Local delivery does not preclude a systemic pharmacological effect.

“Amino” refers to the —NH₂ radical.

“Cyano” or “nitrile” refers to the —CN radical.

“Hydroxy” or “hydroxyl” refers to the —OH radical.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double (alkenyl) and/or triplebonds (alkynyl)), having, for example, from one to twenty-four carbonatoms (C₁-C₂₄ alkyl), four to twenty carbon atoms (C₄-C₂₀ alkyl), six tosixteen carbon atoms (C₆-C₁₆ alkyl), six to nine carbon atoms (C₆-C₉alkyl), one to fifteen carbon atoms (C₁-C₁₅ alkyl), one to twelve carbonatoms (C₁-C₁₂ alkyl), one to eight carbon atoms (C₁-C₈ alkyl) or one tosix carbon atoms (C₁-C₆ alkyl) and which is attached to the rest of themolecule by a single bond, e.g., methyl, ethyl, n propyl, 1 methylethyl(iso propyl), n butyl, n pentyl, 1,1-dimethylethyl (t butyl),3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl,pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl,hexynyl, and the like. Unless stated otherwise specifically in thespecification, an alkyl group is optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, which is saturated orunsaturated (i.e., contains one or more double (alkenylene) and/ortriple bonds (alkynylene)), and having, for example, from one totwenty-four carbon atoms (C₁-C₂₄ alkylene), one to fifteen carbon atoms(C₁-C₁₅ alkylene),one to twelve carbon atoms (C₁-C₁₂ alkylene), one toeight carbon atoms (C₁-C₈ alkylene), one to six carbon atoms (C₁-C₆alkylene), two to four carbon atoms (C₂-C₄ alkylene), one to two carbonatoms (C₁-C₂ alkylene), e.g., methylene, ethylene, propylene,n-butylene, ethenylene, propenylene, n-butenylene, propynylene,n-butynylene, and the like. The alkylene chain is attached to the restof the molecule through a single or double bond and to the radical groupthrough a single or double bond. The points of attachment of thealkylene chain to the rest of the molecule and to the radical group canbe through one carbon or any two carbons within the chain. Unless statedotherwise specifically in the specification, an alkylene chain may beoptionally substituted.

“Alkoxy” refers to a radical of the formula —OR_(a) where R_(a) is analkyl radical as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, an alkoxygroup may be optionally substituted.

“Alkylaminyl” refers to a radical of the formula —NHR_(a) or—NR_(a)R_(a) where each R_(a) is, independently, an alkyl radical asdefined above containing one to twelve carbon atoms. Unless statedotherwise specifically in the specification, an alkylaminyl group isoptionally substituted.

“Aryl” refers to a hydrocarbon ring system radical comprising hydrogen,6 to 18 carbon atoms and at least one aromatic ring. For purposes ofthis invention, the aryl radical may be a monocyclic, bicyclic,tricyclic or tetracyclic ring system, which may include fused or bridgedring systems. Aryl radicals include, but are not limited to, arylradicals derived from aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, fluoranthene, fluorene,as-indacene, s-indacene, indane, indene, naphthalene, phenalene,phenanthrene, pleiadene, pyrene, and triphenylene. “Arylene” is adivalent aryl radical. Unless stated otherwise specifically in thespecification, the term “arylene,” “aryl” or the prefix “ar-” (such asin “aralkyl”) is meant to include aryl radicals that are optionallysubstituted.

A “carbocyclic ring” is a ring wherein each ring atom is carbon.Carbocyclic rings may saturated or unsaturated, including aromaticrings. Unless stated otherwise specifically in the specification, acarbocylic group is optionally substituted.

“Cycloalkyl” or “carbocyclic ring” refers to a stable non-aromaticmonocyclic or polycyclic carbocyclic ring, which may include fused orbridged ring systems, having from three to fifteen carbon atoms,preferably having from three to ten carbon atoms, and which is saturatedor unsaturated and attached to the rest of the molecule by a singlebond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclicradicals include, for example, adamantyl, norbornyl, decalinyl,7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. “Cycloalkylene” is adivalent cycloalkyl radical. Unless otherwise stated specifically in thespecification, cycloalkyl and cycloalkylene groups are optionallysubstituted.

“Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to18-membered (e.g., 5, 6 or 7-membered) non-aromatic ring radical,wherein at least one ring atom is a heteroatom selected from the groupconsisting of nitrogen, oxygen and sulfur, and the remaining ring atomsare selected from the group consisting of carbon nitrogen, oxygen andsulfur. Unless stated otherwise specifically in the specification, theheterocyclyl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heterocyclylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized; and the heterocyclyl radical may be partially or fullysaturated. Examples of such heterocyclyl radicals include, but are notlimited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl,imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl,morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and1,1-dioxo-thiomorpholinyl. “Heterocyclene” is a divalent heterocycleradical. Unless stated otherwise specifically in the specification,heterocyclyl and heterocylene groups are optionally substituted.

“Heteroaryl” refers to a 5- to 14-membered ring system radicalcomprising one to thirteen carbon atoms, one to six heteroatoms selectedfrom the group consisting of nitrogen, oxygen and sulfur, and at leastone aromatic ring. For purposes of this invention, the heteroarylradical may be a monocyclic, bicyclic, tricyclic or tetracyclic ringsystem, which may include fused or bridged ring systems; and thenitrogen, carbon or sulfur atoms in the heteroaryl radical may beoptionally oxidized; the nitrogen atom may be optionally quaternized.Examples include, but are not limited to, azepinyl, acridinyl,benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl,furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl,isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl,isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl,1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl,phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl,quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl,triazinyl, and thiophenyl (i.e. thienyl). “Heteroarylene” is a divalentheteroaryl radical. Unless stated otherwise specifically in thespecification, heteroaryl and heteroarylene groups are optionallysubstituted.

“Fused” refers to any ring structure described herein which is fused toan existing ring structure in the compounds of the invention. When thefused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atomon the existing ring structure which becomes part of the fusedheterocyclyl ring or the fused heteroaryl ring may be replaced with anitrogen atom.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo.

“Hydroxylalkyl” is an alkyl group comprising at least one hydroxylsubstituent. Unless stated otherwise specifically in the specification,hydroxylalkyl groups are optionally substituted.

“Aminoalkyl” is an alkyl group comprising at least one aminosubstituent. Unless stated otherwise specifically in the specification,aminoalkyl groups are optionally substituted.

“Alkylaminylalkyl” is an alkyl group comprising at least one alkylaminylsubstituent. Unless stated otherwise specifically in the specification,alkylaminylalkyl groups are optionally substituted.

“Alkoxylalkyl” is an alkyl group comprising at least one alkoxysubstituent. Unless stated otherwise specifically in the specification,alkoxyalkyl groups are optionally substituted. “Alkoxycarbonyl” isradical of formula —C(═O)R_(a), wherein R_(a) is an alkoxy group. Unlessstated otherwise specifically in the specification, alkoxycarbonylgroups are optionally substituted.

“Alkylcarbonyloxy” is radical of formula —OC(═O)R_(a), wherein R_(a) isan alkyl group. Unless stated otherwise specifically in thespecification, alkylcarbonyloxy groups are optionally substituted.

“Alkylcarbonyloxyalkyl” is radical of formula —R^(b)OC(═O)R_(a), whereinR_(a) is an alkyl group and R^(b) is alkylene. Unless stated otherwisespecifically in the specification, alkylcarbonyloxyalkyl groups areoptionally substituted.

“Alkylcarbonyl” is radical of formula —C(═O)R_(a), wherein R_(a) is analkyl group. Unless stated otherwise specifically in the specification,alkylcarbonyl groups are optionally substituted.

The term “substituted” used herein means any of the above groups (e.g.alkyl, alkylene, alkene, alkenylene, alkyne, alkynylene, alkoxy,alkylaminyl, aryl, arylene, carbocyclic, cycloalkyl, cycloalkylene,heterocyclyl, heterocylene, heteroaryl, heteroarylene, hydroxylalkyl,aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl,alkylcarbonyloxyalkyl or alkylcarbonyl) wherein at least one hydrogenatom is replaced by a bond to a non-hydrogen atom such as, but notlimited to: a halogen atom such as F, Cl, Br, or I; oxo groups (═O);hydroxyl groups (—OH); C₁-C₁₂ alkyl groups; cycloalkyl groups;—(C═O)OR′; —O(C═O)R′; —C(═O)R′; —OR; —S(O)_(x)R′; —S—SR′; —C(═O)SR′;—SC(═O)R′; —NR′R′; —NR′C(═O)R′; —C(═O)NR′R′; ′NR′C(═O)NR′R′;—OC(═O)NR′R′; —NR′C(═O)OR′; —NR′S(O)_(x)NR′R′; —NR′S(O)_(x)R′; and—S(O)_(x)NR′R′, wherein: R′ is, at each occurrence, independently H,C₁-C₁₅ alkyl or cycloalkyl, and x is 0, 1 or 2. In some embodiments thesubstituent is a C₁-C₁₂ alkyl group. In other embodiments, thesubstituent is a cycloalkyl group. In other embodiments, the substituentis a halo group, such as fluoro. In other embodiments, the substituentis an oxo group. In other embodiments, the substituent is a hydroxylgroup. In other embodiments, the substituent is an alkoxy group (—40OR′). In other embodiments, the substituent is a carboxyl group. Inother embodiments, the substituent is an amine group(—NR′R′).

A “polar functional group” is a functional group having at least onepolarized bond. Polar functional groups often comprise at least oneheteroatom (e.g., O, N or S). Polar functional are also typicallyhydrophilic and tend to increase the solubility of the compound in water(as compared to solubility of the same compound with a non-polarfunctional group). Examples of polar functional groups include, but arenot limited to groups containing one or more of the following groups:hydroxyl, alkoxy, carbonyl (i.e., C(═O)), ester (—OC(═O)— and —C(═O)O—),cyano, amide (e.g., —NR_(a)C(═O)(R_(a))— or —C(═O)N(R_(a))₂—, whereineach R_(a) is independently alkyl), alkoxy, ether (e.g., R_(a)—O—R_(a),wherein each R_(a) is independently alkyl), amino, alkylamino,heterocyclyl, heteroaryl, S, S(═O) and S(═O)₂ groups.

“Optional” or “optionally” (e.g., optionally substituted) means that thesubsequently described event of circumstances may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances in which it does not. For example, “optionallysubstituted alkyl” means that the alkyl radical may or may not besubstituted and that the description includes both substituted alkylradicals and alkyl radicals having no substitution.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound of the invention. Thus, the term “prodrug” refers to ametabolic precursor of a compound of the invention that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject in need thereof, but is converted in vivo to an activecompound of the invention. Prodrugs are typically rapidly transformed invivo to yield the parent compound of the invention, for example, byhydrolysis in blood. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in a mammalianorganism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24(Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi,T., et al., A.C.S. Symposium Series, Vol. 14, and in BioreversibleCarriers in Drug Design, Ed. Edward B. Roche, American PharmaceuticalAssociation and Pergamon Press, 1987.

The term “prodrug” is also meant to include any covalently bondedcarriers, which release the active compound of the invention in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of acompound of the invention may be prepared by modifying functional groupspresent in the compound of the invention in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound of the invention. Prodrugs include compounds of theinvention wherein a hydroxy, amino or mercapto group is bonded to anygroup that, when the prodrug of the compound of the invention isadministered to a mammalian subject, cleaves to form a free hydroxy,free amino or free mercapto group, respectively. Examples of prodrugsinclude, but are not limited to, acetate, formate and benzoatederivatives of alcohol or amide derivatives of amine functional groupsin the compounds of the invention and the like.

Embodiments of the invention disclosed herein are also meant toencompass all pharmaceutically acceptable compounds of the compound ofstructure (I) or (II) being isotopically-labelled by having one or moreatoms replaced by an atom having a different atomic mass or mass number.Examples of isotopes that can be incorporated into the disclosedcompounds include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, chlorine, and iodine, such as ²H, ³H, ¹¹C, ¹³C,¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I,respectively. These radiolabeled compounds could be useful to helpdetermine or measure the effectiveness of the compounds, bycharacterizing, for example, the site or mode of action, or bindingaffinity to pharmacologically important site of action. Certainisotopically-labelled compounds of structure (I) or (II), for example,those incorporating a radioactive isotope, are useful in drug and/orsubstrate tissue distribution studies. The radioactive isotopes tritium,i.e., ³H, and carbon-14, i.e., ¹⁴C, are particularly useful for thispurpose in view of their ease of incorporation and ready means ofdetection.

Substitution with heavier isotopes such as deuterium, i.e., ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy. Isotopically-labeled compoundsof structure (I) or (II) can generally be prepared by conventionaltechniques known to those skilled in the art or by processes analogousto those described in the Preparations and Examples as set out belowusing an appropriate isotopically-labeled reagent in place of thenon-labeled reagent previously employed.

Embodiments of the invention disclosed herein are also meant toencompass the in vivo metabolic products of the disclosed compounds.Such products may result from, for example, the oxidation, reduction,hydrolysis, amidation, esterification, and the like of the administeredcompound, primarily due to enzymatic processes. Accordingly, theinvention includes compounds produced by a process comprisingadministering a compound of this invention to a mammal for a period oftime sufficient to yield a metabolic product thereof. Such products aretypically identified by administering a radiolabeled compound of theinvention in a detectable dose to an animal, such as rat, mouse, guineapig, monkey, or to human, allowing sufficient time for metabolism tooccur, and isolating its conversion products from the urine, blood orother biological samples.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Mammal” includes humans and both domestic animals such as laboratoryanimals and household pets (e.g., cats, dogs, swine, cattle, sheep,goats, horses, rabbits), and non-domestic animals such as wildlife andthe like.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals. “Pharmaceutically acceptable salt” includesboth acid and base addition salts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Often crystallizations produce a solvate of the compounds of theinvention (i.e., a compound of structure (I) or (II)). As used herein,the term “solvate” refers to an aggregate that comprises one or moremolecules of a compound of the invention with one or more molecules ofsolvent. The solvent may be water, in which case the solvate may be ahydrate. Alternatively, the solvent may be an organic solvent. Thus, thecompounds of the present invention may exist as a hydrate, including amonohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate,tetrahydrate and the like, as well as the corresponding solvated forms.The compound of the invention may be true solvates, while in othercases, the compound of the invention may merely retain adventitiouswater or be a mixture of water plus some adventitious solvent.

A “pharmaceutical composition” refers to a formulation of a compound ofthe invention and a medium generally accepted in the art for thedelivery of the biologically active compound to mammals, e.g., humans.Such a medium includes all pharmaceutically acceptable carriers,diluents or excipients therefor.

“Effective amount” or “therapeutically effective amount” refers to thatamount of a compound of the invention which, when administered to amammal, preferably a human, is sufficient to effect treatment in themammal, preferably a human. The amount of a lipid nanoparticle of theinvention which constitutes a “therapeutically effective amount” willvary depending on the compound, the condition and its severity, themanner of administration, and the age of the mammal to be treated, butcan be determined routinely by one of ordinary skill in the art havingregard to his own knowledge and to this disclosure.

“Treating” or “treatment” as used herein covers the treatment of thedisease or condition of interest in a mammal, preferably a human, havingthe disease or condition of interest, and includes:

(i) preventing the disease or condition from occurring in a mammal, inparticular, when such mammal is predisposed to the condition but has notyet been diagnosed as having it;

(ii) inhibiting the disease or condition, i.e., arresting itsdevelopment;

(iii) relieving the disease or condition, i.e., causing regression ofthe disease or condition; or

(iv) relieving the symptoms resulting from the disease or condition,i.e., relieving pain without addressing the underlying disease orcondition. As used herein, the terms “disease” and “condition” may beused interchangeably or may be different in that the particular maladyor condition may not have a known causative agent (so that etiology hasnot yet been worked out) and it is therefore not yet recognized as adisease but only as an undesirable condition or syndrome, wherein a moreor less specific set of symptoms have been identified by clinicians.

The compounds of the invention (e.g., compounds of structure (I) or(II)), or their pharmaceutically acceptable salts may contain one ormore asymmetric centers and may thus give rise to enantiomers,diastereomers, and other stereoisomeric forms that may be defined, interms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)-for amino acids. Embodiments of the present invention are meant toinclude all such possible isomers, as well as their racemic andoptically pure forms. Optically active (+) and (−), (R)- and (S)-, or(D)- and (L)- isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques, for example,chromatography and fractional crystallization. Conventional techniquesfor the preparation/isolation of individual enantiomers include chiralsynthesis from a suitable optically pure precursor or resolution of theracemate (or the racemate of a salt or derivative) using, for example,chiral high pressure liquid chromatography (HPLC). When the compoundsdescribed herein contain olefinic double bonds or other centers ofgeometric asymmetry, and unless specified otherwise, it is intended thatthe compounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present invention includestautomers of any said compounds.

Compounds

In an aspect, the invention provides novel lipid compounds which arecapable of combining with other lipid components such as neutral lipids,charged lipids, steroids and/or polymer conjugated-lipids to form lipidnanoparticles with oligonucleotides. Without wishing to be bound bytheory, it is thought that these lipid nanoparticles shieldoligonucleotides from degradation in the serum and provide for effectivedelivery of oligonucleotides to cells in vitro and in vivo.

In one embodiment, the compounds have the following structure (I):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

one of G¹ or G² is, at each occurrence, —O(C═O)—, —(C═O)O—, —C(═O)—,—O—, —S(O)_(y−), —S—S—, —C(═O)S—, SC(═O)—, —N(R^(a))C(═O)—,—C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—, —OC(═O)N(R^(a))— or—N(R^(a))C(═O)O—, and the other of G¹ or G² is, at each occurrence,—O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(y−), —S—S—, —C(═O)S—, —SC(═O)—,—N(R^(a))C(═O)—, —C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—,—OC(═O)N(R^(a))— or —N(R^(a))C(═O)O— or a direct bond;

L is, at each occurrence, ˜O(C═O)—, wherein ˜ represents a covalent bondto X;

X is CR^(a);

Z is alkyl, cycloalkyl or a monovalent moiety comprising at least onepolar functional group when n is 1; or Z is alkylene, cycloalkylene or apolyvalent moiety comprising at least one polar functional group when nis greater than 1;

R^(a) is, at each occurrence, independently H, C₁-C₁₂ alkyl, C₁-C₁₂hydroxylalkyl, C₁-C₁₂ aminoalkyl, C₁-C₁₂ alkylaminylalkyl, C₁-C₁₂alkoxyalkyl, C₁-C₁₂ alkoxycarbonyl, C₁-C₁₂ alkylcarbonyloxy, C₁-C₁₂alkylcarbonyloxyalkyl or C₁-C₁₂ alkylcarbonyl;

R is, at each occurrence, independently either: (a) H or C₁-C₁₂ alkyl;or (b) R together with the carbon atom to which it is bound is takentogether with an adjacent R and the carbon atom to which it is bound toform a carbon-carbon double bond;

R¹ and R² have, at each occurrence, the following structure,respectively:

a¹ and a² are, at each occurrence, independently an integer from 3 to12;

b¹ and b² are, at each occurrence, independently 0 or 1;

c¹ and c² are, at each occurrence, independently an integer from 5 to10;

d¹ and d² are, at each occurrence, independently an integer from 5 to10;

y is, at each occurrence, independently an integer from 0 to 2; and

n is an integer from 1 to 6,

wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl,alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy,alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted withone or more substituent.

In some embodiments of structure (I), G¹ and G² are each independently—O(C═O)— or —(C═O)O—.

In other embodiments of structure (I), X is CH.

In different embodiments of structure (I), the sum of a¹+b ¹+c¹ or thesum of a²+b²+c² is an integer from 12 to 26.

In still other embodiments of structure (I), a¹ and a² are independentlyan integer from 3 to 10. For example, in some embodiments a¹ and a² areindependently an integer from 4 to 9.

In various embodiments of structure (I), b¹ and b² are 0. In differentembodiments, b¹ and b² are 1.

In more embodiments of structure (I), c¹, c², d¹ and d² areindependently an integer from 6 to 8.

In other embodiments of structure (I), c¹ and c² are, at eachoccurrence, independently an integer from 6 to 10, and d¹ and d² are, ateach occurrence, independently an integer from 6 to 10.

In other embodiments of structure (I), c¹ and c² are, at eachoccurrence, independently an integer from 5 to 9, and d¹ and d² are, ateach occurrence, independently an integer from 5 to 9.

In more embodiments of structure (I), Z is alkyl, cycloalkyl or amonovalent moiety comprising at least one polar functional group when nis 1. In other embodiments, Z is alkyl.

In various embodiments of the foregoing, R is, at each occurrence,independently either: (a) H or methyl; or (b) R together with the carbonatom to which it is bound is taken together with an adjacent R and thecarbon atom to which it is bound to form a carbon-carbon double bond. Incertain embodiments, each R is H. In other embodiments at least one Rtogether with the carbon atom to which it is bound is taken togetherwith an adjacent R and the carbon atom to which it is bound to form acarbon-carbon double bond.

In other embodiments of the compound of structure (I), R¹ and R²independently have one of the following structures:

In certain embodiments of structure (I), the compound has one of thefollowing structures:

In still different embodiments is provided a compound having thefollowing structure (II):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

one of G¹ or G² is, at each occurrence, —O(C═O)—, —(C═O)O—, —C(═O)—,—O—, —S(O)_(y−), —S—S—, —C(═O)S—, SC(═O)—, —N(R^(a))C(═O)—,—C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—, —OC(═O)N(R^(a))— or—N(R^(a))C(═O)O—, and the other of G¹ or G² is, at each occurrence,—O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(y−), —S—S—, —C(═O)S—, —SC(═O)—,—N(R^(a))C(═O)—, —C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—,—OC(═O)N(R^(a))— or —N(R^(a))C(═O)O— or a direct bond;

L is, at each occurrence, ˜O(C═O)—, wherein ˜ represents a covalent bondto X;

X is CR^(a);

Z is alkyl, cycloalkyl or a monovalent moiety comprising at least onepolar functional group when n is 1; or Z is alkylene, cycloalkylene or apolyvalent moiety comprising at least one polar functional group when nis greater than 1;

R^(a) is, at each occurrence, independently H, C₁-C₁₂ alkyl, C₁-C₁₂hydroxylalkyl, C₁-C₁₂ aminoalkyl, C₁-C₁₂ alkylaminylalkyl, C₁-C₁₂alkoxyalkyl, C₁-C₁₂ alkoxycarbonyl, C₁-C₁₂ alkylcarbonyloxy, C₁-C₁₂alkylcarbonyloxyalkyl or C₁-C₁₂ alkylcarbonyl;

R is, at each occurrence, independently either: (a) H or C₁-C₁₂ alkyl;or (b) R together with the carbon atom to which it is bound is takentogether with an adjacent R and the carbon atom to which it is bound toform a carbon-carbon double bond;

R¹ and R² have, at each occurrence, the following structure,respectively:

R′ is, at each occurrence, independently H or C₁-C₁₂ alkyl;

a¹ and a² are, at each occurrence, independently an integer from 3 to12;

b¹ and b² are, at each occurrence, independently 0 or 1;

c¹ and c² are, at each occurrence, independently an integer from 2 to12;

d¹ and d² are, at each occurrence, independently an integer from 2 to12;

y is, at each occurrence, independently an integer from 0 to 2; and

n is an integer from 1 to 6,

wherein a¹, a², c¹, c², d¹ and d² are selected such that the sum ofa¹+c¹+d¹ is an integer from 18 to 30, and the sum of a²+c²+d² is aninteger from 18 to 30, and wherein each alkyl, alkylene, hydroxylalkyl,aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl,alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionallysubstituted with one or more substituent.

In certain embodiments of structure (II), G¹ and G² are eachindependently

—O(C═O)— or —(C═O)O—.

In other embodiments of structure (II), X is CH.

In some embodiments of structure (II), the sum of a¹+c¹+d¹ is an integerfrom 20 to 30, and the sum of a²+c²+d² is an integer from 18 to 30. Inother embodiments, the sum of a¹+c¹+d¹ is an integer from 20 to 30, andthe sum of a²+c²+d² is an integer from 20 to 30. In more embodiments ofstructure (II), the sum of a¹+b¹+c¹ or the sum of a²+b²+c² is an integerfrom 12 to 26. In other embodiments, a¹, a², c¹, c², d¹ and d² areselected such that the sum of a¹+c¹+d¹ is an integer from 18 to 28, andthe sum of a²+c²+d² is an integer from 18 to 28,

In still other embodiments of structure (II), a¹ and a² areindependently an integer from 3 to 10, for example an integer from 4 to9.

In yet other embodiments of structure (II), b¹ and b² are 0. Indifferent embodiments b¹ and b² are 1.

In certain other embodiments of structure (II), c¹, c², d¹ and d² areindependently an integer from 6 to 8.

In different other embodiments of structure (II), Z is alkyl or amonovalent moiety comprising at least one polar functional group when nis 1; or Z is alkylene or a polyvalent moiety comprising at least onepolar functional group when n is greater than 1.

In more embodiments of structure (II), Z is alkyl, cycloalkyl or amonovalent moiety comprising at least one polar functional group when nis 1. In other embodiments, Z is alkyl.

In other different embodiments of structure (II), R is, at eachoccurrence, independently either: (a) H or methyl; or (b) R togetherwith the carbon atom to which it is bound is taken together with anadjacent R and the carbon atom to which it is bound to form acarbon-carbon double bond. For example in some embodiments each R is H.In other embodiments at least one R together with the carbon atom towhich it is bound is taken together with an adjacent R and the carbonatom to which it is bound to form a carbon-carbon double bond.

In more embodiments, each R′ is H.

In certain embodiments of structure (II), the sum of a¹+c¹+d¹ is aninteger from 20 to 25, and the sum of a²+c²+d² is an integer from 20 to25.

In other embodiments of structure (II), R¹ and R² independently have oneof the following structures:

In more embodiments of structure (II), the compound has one of thefollowing structures:

In any of the foregoing embodiments, n is 1. In other of the foregoingembodiments, n is greater than 1.

In more of any of the foregoing embodiments, Z is a mono- or polyvalentmoiety comprising at least one polar functional group. In someembodiments, Z is a monovalent moiety comprising at least one polarfunctional group. In other embodiments, Z is a polyvalent moietycomprising at least one polar functional group.

In more of any of the foregoing embodiments, the polar functional groupis a hydroxyl, alkoxy, ester, cyano, amide, amino, alkylaminyl,heterocyclyl or heteroaryl functional group.

In any of the foregoing embodiments, Z is hydroxyl, hydroxylalkyl,alkoxyalkyl, amino, aminoalkyl, alkylaminyl, alkylaminylalkyl,heterocyclyl or heterocyclylalkyl.

In some other embodiments, Z has the following structure:

wherein:

-   -   R⁵ and R⁶ are independently H or C₁-C₆ alkyl;

R⁷ and R⁸ are independently H or C₁-C₆ alkyl or R⁷ and R⁸, together withthe nitrogen atom to which they are attached, join to form a 3-7membered heterocyclic ring; and

x is an integer from 0 to 6.

In still different embodiments, Z has the following structure:

wherein:

R⁵ and R⁶ are independently H or C₁-C₆ alkyl;

R⁷ and R⁸ are independently H or C₁-C₆ alkyl or R⁷ and R⁸, together withthe nitrogen atom to which they are attached, join to form a 3-7membered heterocyclic ring; and

x is an integer from 0 to 6.

In still different embodiments, Z has the following structure:

wherein:

R⁵ and R⁶ are independently H or C₁-C₆ alkyl; R⁷ and R⁸ areindependently H or C₁-C₆ alkyl or R⁷ and R⁸, together with the nitrogenatom to which they are attached, join to form a 3-7 memberedheterocyclic ring; and

x is an integer from 0 to 6.

In some other embodiments, Z is hydroxylalkyl, cyanoalkyl or an alkylsubstituted with one or more ester or amide groups.

For example, in any of the foregoing embodiments, Z has one of thefollowing structures:

In other embodiments, Z-L has one of the following structures:

In other embodiments, Z-L has one of the following structures:

In still other embodiments, X is CH and Z-L has one of the followingstructures:

In various different embodiments, the compound has one of the structuresset forth in Table 1 below.

TABLE 1 Representative Compounds No. Structure 1

2

3

In other embodiments, the invention does not include any of thecompounds illustrated in Table 1.

It is understood that any embodiment of the compounds of structure (I)or (II), as set forth above, and any specific substituent and/orvariable in the compound structure (I) or (II), as set forth above, maybe independently combined with other embodiments and/or substituentsand/or variables of compounds of structure (I) or (II) to formembodiments of the inventions not specifically set forth above. Inaddition, in the event that a list of substituents and/or variables islisted for any particular R group, L group, G group, Z group, orvariables a, n, x, y, or z in a particular embodiment and/or claim, itis understood that each individual substituent and/or variable may bedeleted from the particular embodiment and/or claim and that theremaining list of substituents and/or variables will be considered to bewithin the scope of the invention.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

In some embodiments, compositions comprising any one or more of thecompounds of structure (I) or (II) and a therapeutic agent are provided.For example, in some embodiments, the compositions comprise any of thecompounds of structure (I) or (II) and a therapeutic agent and one ormore excipient selected from neutral lipids, steroids and polymerconjugated lipids. Other pharmaceutically acceptable excipients and/orcarriers are also included in various embodiments of the compositions.

In some embodiments, the neutral lipid is selected from DSPC, DPPC,DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid isDSPC. In various embodiments, the molar ratio of the compound to theneutral lipid ranges from about 2:1 to about 8:1.

In various embodiments, the compositions further comprise a steroid orsteroid analogue. In certain embodiments, the steroid or steroidanalogue is cholesterol. In some of these embodiments, the molar ratioof the compound to cholesterol ranges from about 5:1 to 1:1.

In various embodiments, the polymer conjugated lipid is a pegylatedlipid. For example, some embodiments include a pegylated diacylglycerol(PEG-DAG) such as1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), apegylated phosphatidylethanoloamine (PEG-PE), a PEG succinatediacylglycerol (PEG-S-DAG) such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate(PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEGdialkoxypropylcarbamate such asω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. Invarious embodiments, the molar ratio of the compound to the pegylatedlipid ranges from about 100:1 to about 20:1.

In some embodiments, the composition comprises a pegylated lipid havingthe following structure (III):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:

R³ and R⁴ are each independently a straight or branched, saturated orunsaturated alkyl chain containing from 10 to 30 carbon atoms, whereinthe alkyl chain is optionally interrupted by one or more ester bonds;and

w has a mean value ranging from 30 to 60.

In some embodiments, R³ and R⁴ are each independently straight,saturated alkyl chains containing from 12 to 16 carbon atoms. In otherembodiments, the average w is about 45. In other embodiments, theaverage w ranges from 42 to 55.

For example, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55.In some specific embodiments, w is about 49.

In some embodiments of the foregoing composition, the therapeutic agentcomprises a nucleic acid. For example, in some embodiments, the nucleicacid is selected from antisense, plasmid DNA, and messenger RNA.

In other different embodiments, the invention is directed to a methodfor administering a therapeutic agent to a patient in need thereof, themethod comprising preparing or providing any of the foregoingcompositions and administering the composition to the patient.

For the purposes of administration, the compounds of the presentinvention (typically in the form of lipid nanoparticles in combinationwith a therapeutic agent) may be administered as a raw chemical or maybe formulated as pharmaceutical compositions. Pharmaceuticalcompositions of the present invention comprise a compound of structure(I) or (II) and one or more pharmaceutically acceptable carrier, diluentor excipient. The compound of structure (I) or (II) is present in thecomposition in an amount which is effective to form a lipid nanoparticleand deliver the therapeutic agent, e.g., for treating a particulardisease or condition of interest. Appropriate concentrations and dosagescan be readily determined by one skilled in the art.

Administration of the compositions of the invention can be carried outvia any of the accepted modes of administration of agents for servingsimilar utilities. The pharmaceutical compositions of the invention maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suspensions, suppositories, injections, inhalants, gels,microspheres, and aerosols. Typical routes of administering suchpharmaceutical compositions include, without limitation, oral, topical,transdermal, inhalation, parenteral, sublingual, buccal, rectal,vaginal, and intranasal. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intradermal,intrasternal injection or infusion techniques. Pharmaceuticalcompositions of the invention are formulated so as to allow the activeingredients contained therein to be bioavailable upon administration ofthe composition to a patient. Compositions that will be administered toa subject or patient take the form of one or more dosage units, wherefor example, a tablet may be a single dosage unit, and a container of acompound of the invention in aerosol form may hold a plurality of dosageunits. Actual methods of preparing such dosage forms are known, or willbe apparent, to those skilled in this art; for example, see Remington:The Science and Practice of Pharmacy, 20th Edition (Philadelphia Collegeof Pharmacy and Science, 2000). The composition to be administered will,in any event, contain a therapeutically effective amount of a compoundof the invention, or a pharmaceutically acceptable salt thereof, fortreatment of a disease or condition of interest in accordance with theteachings of this invention.

A pharmaceutical composition of the invention may be in the form of asolid or liquid. In one aspect, the carrier(s) are particulate, so thatthe compositions are, for example, in tablet or powder form. Thecarrier(s) may be liquid, with the compositions being, for example, anoral syrup, injectable liquid or an aerosol, which is useful in, forexample, inhalatory administration.

When intended for oral administration, the pharmaceutical composition ispreferably in either solid or liquid form, where semi-solid,semi-liquid, suspension and gel forms are included within the formsconsidered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like form. Such a solidcomposition will typically contain one or more inert diluents or ediblecarriers. In addition, one or more of the following may be present:binders such as carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, gum tragacanth or gelatin; excipients suchas starch, lactose or dextrins, disintegrating agents such as alginicacid, sodium alginate, Primogel, corn starch and the like; lubricantssuch as magnesium stearate or Sterotex; glidants such as colloidalsilicon dioxide; sweetening agents such as sucrose or saccharin; aflavoring agent such as peppermint, methyl salicylate or orangeflavoring; and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, forexample, a gelatin capsule, it may contain, in addition to materials ofthe above type, a liquid carrier such as polyethylene glycol or oil.

The pharmaceutical composition may be in the form of a liquid, forexample, an elixir, syrup, solution, emulsion or suspension. The liquidmay be for oral administration or for delivery by injection, as twoexamples. When intended for oral administration, preferred compositioncontain, in addition to the present compounds, one or more of asweetening agent, preservatives, dye/colorant and flavor enhancer. In acomposition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of the invention, whether they besolutions, suspensions or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose; agents to act as cryoprotectants such assucrose or trehalose. The parenteral preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition of the invention intended for eitherparenteral or oral administration should contain an amount of a compoundof the invention such that a suitable dosage will be obtained.

The pharmaceutical composition of the invention may be intended fortopical administration, in which case the carrier may suitably comprisea solution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device.

The pharmaceutical composition of the invention may be intended forrectal administration, in the form, for example, of a suppository, whichwill melt in the rectum and release the drug. The composition for rectaladministration may contain an oleaginous base as a suitablenonirritating excipient. Such bases include, without limitation,lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition of the invention may include variousmaterials, which modify the physical form of a solid or liquid dosageunit. For example, the composition may include materials that form acoating shell around the active ingredients. The materials that form thecoating shell are typically inert, and may be selected from, forexample, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule.

The pharmaceutical composition of the invention in solid or liquid formmay include an agent that binds to the compound of the invention andthereby assists in the delivery of the compound. Suitable agents thatmay act in this capacity include a monoclonal or polyclonal antibody, ora protein.

The pharmaceutical composition of the invention may consist of dosageunits that can be administered as an aerosol. The term aerosol is usedto denote a variety of systems ranging from those of colloidal nature tosystems consisting of pressurized packages. Delivery may be by aliquefied or compressed gas or by a suitable pump system that dispensesthe active ingredients. Aerosols of compounds of the invention may bedelivered in single phase, bi-phasic, or tri-phasic systems in order todeliver the active ingredient(s). Delivery of the aerosol includes thenecessary container, activators, valves, subcontainers, and the like,which together may form a kit. One skilled in the art, without undueexperimentation may determine preferred aerosols.

The pharmaceutical compositions of the invention may be prepared bymethodology well known in the pharmaceutical art. For example, apharmaceutical composition intended to be administered by injection canbe prepared by combining the lipid nanoparticles of the invention withsterile, distilled water or other carrier so as to form a solution. Asurfactant may be added to facilitate the formation of a homogeneoussolution or suspension. Surfactants are compounds that non-covalentlyinteract with the compound of the invention so as to facilitatedissolution or homogeneous suspension of the compound in the aqueousdelivery system.

The compositions of the invention, or their pharmaceutically acceptablesalts, are administered in a therapeutically effective amount, whichwill vary depending upon a variety of factors including the activity ofthe specific therapeutic agent employed; the metabolic stability andlength of action of the therapeutic agent; the age, body weight, generalhealth, sex, and diet of the patient; the mode and time ofadministration; the rate of excretion; the drug combination; theseverity of the particular disorder or condition; and the subjectundergoing therapy.

Compositions of the invention may also be administered simultaneouslywith, prior to, or after administration of one or more other therapeuticagents. Such combination therapy includes administration of a singlepharmaceutical dosage formulation of a composition of the invention andone or more additional active agents, as well as administration of thecomposition of the invention and each active agent in its own separatepharmaceutical dosage formulation. For example, a composition of theinvention and the other active agent can be administered to the patienttogether in a single oral dosage composition such as a tablet orcapsule, or each agent administered in separate oral dosageformulations. Where separate dosage formulations are used, the compoundsof the invention and one or more additional active agents can beadministered at essentially the same time, i.e., concurrently, or atseparately staggered times, i.e., sequentially; combination therapy isunderstood to include all these regimens.

Preparation methods for the above compounds and compositions aredescribed herein below and/or known in the art.

It will be appreciated by those skilled in the art that in the processdescribed herein the functional groups of intermediate compounds mayneed to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

It will also be appreciated by those skilled in the art, although suchprotected derivatives of compounds of this invention may not possesspharmacological activity as such, they may be administered to a mammaland thereafter metabolized in the body to form compounds of theinvention which are pharmacologically active. Such derivatives maytherefore be described as “prodrugs”. All prodrugs of compounds of thisinvention are included within the scope of the invention.

Furthermore, all compounds of the invention which exist in free base oracid form can be converted to their pharmaceutically acceptable salts bytreatment with the appropriate inorganic or organic base or acid bymethods known to one skilled in the art. Salts of the compounds of theinvention can be converted to their free base or acid form by standardtechniques.

The disclosed compounds can be prepared according to the followingGeneral Reaction Scheme I and methods analogous to those disclosed inPCT Pub. No. WO 2013/086322, the full disclosure of which is herebyincorporated by reference in its entirety. It is understood that oneskilled in the art may be able to make these compounds by similarmethods or by combining other methods known to one skilled in the art.It is also understood that one skilled in the art would be able to make,in a similar manner as described below, other compounds of structure (I)or (II) not specifically illustrated below by using the appropriatestarting components and modifying the parameters of the synthesis asneeded. In general, starting components may be obtained from sourcessuch as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, MatrixScientific, TCI, and Fluorochem USA, etc. or synthesized according tosources known to those skilled in the art (see, for example, AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure, 5th edition(Wiley, December 2000)) or prepared as described in this invention.

General Reaction Scheme 1 (“Method A”) provides a method for preparationof exemplary compounds of structure (I) or (II) (i.e., compound “G”),wherein R, R¹, a¹, a² and Z are as defined herein, and PG is an alcoholprotecting group such as tetrahydropyran. Compounds of structure A arepurchased or prepared according to methods known in the art. Reaction ofA with ethyl formate B under Grignard conditions yields alcohol C, whichcan then be coupled with acid D under standard conditions to yield E.Removal of the protecting group followed by coupling with acid F yieldsG.

General Reaction Scheme 2 (“Method B”) provides an alternative methodfor preparation of exemplary compounds of structure (I) or (II) (i.e.,compound “P”), wherein R, R¹, a¹, a² and Z are as defined herein and PGis an alcohol protecting group such as tetrahydropyran. Compounds ofstructure H are purchased or prepared according to methods known in theart. The hydroxyl group of Compound H is protected using methods knownin the art (e.g. pyridinium p-toluenesulfonate, dihydropyran) to yieldI. Reaction of I with ethyl formate J under Grignard conditions (e.g.with Mg, I₂) yields alcohol K. The hydroxyl group of compound K can beoxidized (e.g. with pyridinium chlorochromate) and the protecting groupsremoved (e.g. with pyridinium p-toluenesulfonate) to yield compound L.The free hydroxyl groups of L are then coupled with acid M understandard ester coupling conditions to yield N. The carbonyl of N is thenreduced using methods known in the art (e.g. NaBH₄) followed by couplingwith acid O (e.g. with DMAP, EDC.Cl) to yield the desired product P.

General Reaction Scheme 3 (“Method C”) provides another alternativemethod for preparation of exemplary compounds of structure (I) or (II)(i.e., compound “V”), wherein R, R¹, a¹, a² and Z are as defined herein.Compounds of structure Q are purchased or prepared according to methodsknown in the art. Compound Q is used to form R under appropriateconditions (e.g. diethyl acetone dicarboxylate, EtONa). Alcohol S isthen coupled to R using standard conditions (e.g. DMAP, EDC.HCl) toyield T. The carbonyl of T is reduced (e.g. with NaBH₄) followed bycoupling with acid U (e.g. with DMAP, EDC.HCl) to yield the desiredproduct V.

It should be noted that various alternative strategies for preparationof compounds of structure (I) or (II) are available to those of ordinaryskill in the art. For example, other compounds of structure (I) or (II)wherein G¹ and G² are other than ester can be prepared according toanalogous methods using the appropriate starting material. Further, theGeneral Reaction Schemes above depict preparation of a compound ofstructure (I) or (II), wherein R¹ and R² as well as a¹ and a² are thesame; however, this is not a required aspect of the invention andmodifications to the above reaction scheme are possible to yieldcompounds wherein R¹ and R² as well as a¹ and a² are different (i.e.resulting in an asymmetric compound). The use of protecting groups asneeded and other modification to the above General Reaction Schemes willbe readily apparent to one of ordinary skill in the art.

The following examples are provided for purpose of illustration and notlimitation.

EXAMPLE 1 Luciferase mRNA In Vivo Evaluation Using the LipidNanoparticle Compositions

Cationic lipid (MC3), DSPC, cholesterol and PEG-lipid were solubilizedin ethanol at a molar ratio of 50:10:38.5:1.5. Lipid nanoparticles (LNP)were prepared at a total lipid to mRNA weight ratio of approximately10:1 to 30:1. Briefly, the mRNA was diluted to 0.2 mg/mL in 10 to 50 mMcitrate buffer, pH 4. Syringe pumps were used to mix the ethanolic lipidsolution with the mRNA aqueous solution at a ratio of about 1:5 to 1:3(vol/vol) with total flow rates above 15 mL/min. The ethanol was thenremoved and the external buffer replaced with PBS by dialysis. Finally,the lipid nanoparticles were filtered through a 0.2 μm pore sterilefilter. Lipid nanoparticle particle size was approximately 55-95 nmdiameter, and in some instances approximately 70-90 nm diameter asdetermined by quasi-elastic light scattering using a Malvern ZetasizerNano ZS (Malvern, UK) or by using a Nicomp 370 submicron particle sizer(Santa Barbara, Calif.).

Alternatively, Cationic lipid, DSPC, cholesterol and PEG-lipid aresolubilized in ethanol at a molar ratio of at a molar ratio ofapproximately 47.5:10:40.8:1.7, and the LNPs are prepared as set forthabove.

Studies were performed in 6-8 week old female C57BL/6 mice (CharlesRiver) 8-10 week old CD-1 (Harlan) mice (Charles River) according toguidelines established by an institutional animal care committee (ACC)and the Canadian Council on Animal Care (CCAC). Varying doses ofmRNA-lipid nanoparticle were systemically administered by tail veininjection and animals euthanized at specific time points (1, 2, 4, 8 and24 hrs) post-administration. Liver and spleen were collected inpre-weighed tubes, weights determined, immediately snap frozen in liquidnitrogen and stored at −80° C. until processing for analysis.

For liver, approximately 50 mg was dissected for analyses in a 2 mLFastPrep tubes (MP Biomedicals, Solon Ohio). ¼″ ceramic sphere (MPBiomedicals) was added to each tube and 500 μL of Glo Lysis Buffer—GLB(Promega, Madison Wis.) equilibrated to room temperature was added toliver tissue. Liver tissues were homogenized with the FastPrep24instrument (MP Biomedicals) at 2×6.0 m/s for 15 seconds. Homogenate wasincubated at room temperature for 5 minutes prior to a 1:4 dilution inGLB and assessed using SteadyGlo Luciferase assay system (Promega).Specifically, 50 μL of diluted tissue homogenate was reacted with 50 μLof SteadyGlo substrate, shaken for 10 seconds followed by 5 minuteincubation and then quantitated using a CentroXS³ LB 960 luminometer(Berthold Technologies, Germany). The amount of protein assayed wasdetermined by using the BCA protein assay kit (Pierce, Rockford Ill.).Relative luminescence units (RLU) were then normalized to total μgprotein assayed. To convert RLU to ng luciferase a standard curve wasgenerated with QuantiLum Recombinant Luciferase (Promega). Based on thedata provided in FIG. 1, the four-hour time point was chosen forefficacy evaluation of the lipid formulations (see Example 3).

The FLuc mRNA (L-6107) from Trilink Biotechnologies will express aluciferase protein, originally isolated from the firefly, Photinuspyralis. FLuc is commonly used in mammalian cell culture to measure bothgene expression and cell viability. It emits bioluminescence in thepresence of the substrate, luciferin. This capped and polyadenylatedmRNA is fully substituted with 5-methylcytidine and pseudouridine.

EXAMPLE 2 Determination of pK_(A) of Formulated Lipids

As described elsewhere, the pK_(a) of formulated cationic lipids iscorrelated with the effectiveness of LNPs for delivery of nucleic acids(see Jayaraman et al, Angewandte Chemie, International Edition (2012),51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176(2010)). The preferred range of pK_(a) is ˜5 to ˜7. The pK_(a) of eachcationic lipid was determined in lipid nanoparticles using an assaybased on fluorescence of 2-(p-toluidino)-6-napthalene sulfonic acid(TNS). Lipid nanoparticles comprising of cationiclipid/DSPC/cholesterol/PEG-lipid (50/10/38.5/1.5 mol %) in PBS at aconcentration of 0.4 mM total lipid are prepared using the in-lineprocess as described in Example 1. TNS was prepared as a 100 μM stocksolution in distilled water. Vesicles were diluted to 24 μM lipid in 2mL of buffered solutions containing 10 mM HEPES, 10 mM MES, 10 mMammonium acetate, 130 mM NaCl, where the pH ranged from 2.5 to 11. Analiquot of the TNS solution was added to give a final concentration of 1μM and following vortex mixing fluorescence intensity was measured atroom temperature in a SLM Aminco Series 2 Luminescence Spectrophotometerusing excitation and emission wavelengths of 321 nm and 445 nm. Asigmoidal best fit analysis was applied to the fluorescence data and thepK_(a) was measured as the pH giving rise to half-maximal fluorescenceintensity (see FIG. 2).

EXAMPLE 3 Determination of Efficacy of Lipid Nanoparticle FormulationsContaining Various Cationic Lipids Using an In Vivo Luciferase mRNAExpression Rodent Model

The cationic lipids shown in Table 2 have previously been tested withnucleic acids. For comparative purposes, these lipids were also used toformulate lipid nanoparticles containing the FLuc mRNA (L-6107) using anin line mixing method, as described in Example 1 and in PCT/US10/22614,which is hereby incorporated by reference in its entirety. Lipidnanoparticles were formulated using the following molar ratio: 50%Cationic lipid/10% distearoylphosphatidylcholine (DSPC)/38.5%Cholesterol/1.5% PEG lipid (“PEG-DMG”, i.e.,1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol, with anaverage PEG molecular weight of 2000). In alternate embodiments,cationic lipid, DSPC, cholesterol and PEG-lipid are formulated at amolar ratio of approximately 47.5:10:40.8:1.7. Relative activity wasdetermined by measuring luciferase expression in the liver 4 hoursfollowing administration via tail vein injection as described inExample 1. The activity was compared at a dose of 0.3 and 1.0 mg mRNA/kgand expressed as ng luciferase/g liver measured 4 hours afteradministration, as described in Example 1.

TABLE 2 Comparator Lipids showing activity with mRNA Liver Liver Luc @Luc @ 0.3 mg/ 1.0 mg/ Compound kg dose kg dose Structure MC2 4 ± 1 N/D

DLinDMA 13 ± 3  67 ± 20

MC4 41 ± 10 N/D

XTC2 80 ± 28 237 ± 99 

MC3 198 ± 126 757 ± 528

319 (2% PEG) 258 ± 67  681 ± 203

137 281 ± 203 588 ± 303

A 77 ± 40 203 ± 122

Representative compounds of the invention shown in Table 3 wereformulated using the following molar ratio: 50% cationic lipid/10%distearoylphosphatidylcholine (DSPC)/38.5% Cholesterol/1.5% PEG lipid(“PEG-DMA”2-[2-(ω-methoxy(polyethyleneglycol₂₀₀₀)ethoxy]-N,N-ditetradecylacetamide).Relative activity was determined by measuring luciferase expression inthe liver 4 hours following administration via tail vein injection asdescribed in Example 1. The activity was compared relative to activityof MC3 (MC3 activity=1)

TABLE 3 Novel Cationic lipids and Relative Activity Activity RelativeNo. to MC3 Structure 1 ~2

2 ~0.75

3 ~8

EXAMPLE 4 Synthesis of10-{[4′-(Dimethylamino)Butanoyl]Oxy}-19-[(2-Hexyldecanoyl)Oxy]Nonadecyl2-Hexyldecanoate (Compound 1)

10-{[4′-(dimethylamino)butanoyl]oxy}-19-[(2-hexyldecanoyl)oxy]nonadecyl2-hexyldecanoate. A solution of10-[4′-(dimethylamino)butanoyl]oxynonadecan-1,19-diol (0.30 g),2-hexyldecanoic acid (1.1 g),N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (0.42 g) and4-dimethylaminopyridine (0.27 g) in dichloromethane (20 mL) was stirredat room temperature overnight. The solution was washed with dilutedhydrochloric acid, dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel (20 g)column with 0-8% methanol/dichloromethane. The desired product wasafforded (0.42 g)

EXAMPLE 5 Synthesis of1,25-Di-(2′-Hexyldecyl)13-{[4-(Dimethylamino)Butanoyl]Oxy}Pentacosanedioate(Compound 2)

13-oxo-pentacosane-1,25-dioic acid. Sodium ethoxide (1.56 g) wasdissolved in absolute ethanol (30 mL). Diethylacetone dicarboxylate (4.5g) was added and the solution heated to reflux. Ethyl11-bromododecanoate (6.8 g) was slowly added and the solution refluxedfor an hour. Sodium ethoxide (1.53 g) was added, followed by ethyl11-bromododecanoate (18 g). The solution was refluxed overnight. Thereaction mixture was cooled, diluted with water, acidified with dilutehydrochloric acid, and extracted with methylene chloride. The organicfraction was washed with water and the solvent removed. The crudeproduct was passed down a silica gel column (80 g) usingmethanol/methylene chloride to recover unreacted starting materials. Theresidue containing the product was treated with acetic acid (10 mL) andconcentrated hydrochloric acid (20 mL), and then refluxed overnight. Thesolution was cooled, diluted with water and filtered. The collectedprecipitate was recrystallized from acetone, affording the desiredproduct as a white powder (2.9 g).

1,25-di-(2′-hexyldecyl) 13-oxo-pentacosanedioate. A solution of13-oxo-pentacosane-1,25-dioic acid (0.91 g), 4-dimethylaminopyridine(1.1 g), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride(1.0 g) and 2-hexyldecan-1-ol (2.4 g) in dichloromethane (40 mL) wasstirred at room temperature overnight. The solution was washed withdiluted hydrochloric acid, dried over anhydrous magnesium sulfate,filtered and the solvent removed. The residue was passed down a silicagel (20 g) column using 0-4% ethyl acetate/hexane, affording the desiredproduct (1 g).

1,25-di-(2′-hexyldecyl) 13-hydroxy-pentacosanedioate. A solution of1,25-di-(2′-hexyldecyl) 13-oxo-pentacosanedioate (1 g) intetrahydrofuran (10 mL) and methanol (10 mL) was treated with sodiumborohydride (0.18 g). The reaction was stirred for 30 minutes and thendiluted with water, acidified and extracted with dichloromethane. Theorganic fraction was dried over anhydrous magnesium sulfate, filtered,and the solvent removed to afford the crude product (0.95 g). The crudeproduct was used in the next synthetic step without furtherpurification.

1,25-di-(2′-hexyldecyl)13-{[4-(dimethylamino)butanoyl]oxy}pentacosanedioate(Compound 2). A solution of crude 1,25-di-(2′-hexyldecyl)13-hydroxy-pentacosanedioate (0.95 g), 4-dimethylaminopyridine (0.42 g),N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (0.34 g) andN,N-dimethylaminobutyric acid hydrochloride (0.59 g) in dichloromethane(15 mL) was stirred at room temperature for one hour. The solution waswashed with diluted hydrochloric acid followed by aqueous sodiumbicarbonate. The organic phase was dried over anhydrous magnesiumsulfate, filtered, and the solvent removed. The residue was passed downa silica gel (20 g) column using 0-6% methanol/dichloromethane to affordthe desired product (0.81 g).

EXAMPLE 6 Synthesis of10-{[5-(Dimethylamino)Pentanoyl]Oxy}-19-[(2-Hexyldecanoyl)Oxy]Nonadecyl2-Hexyldecanoate (Compound 3)

9-Tetrahydropyranyloxynonan-1-yl bromide. A solution of9-bromononan-1-ol (25.6 g) and dihydropyran (10.5 g) in dichloromethane(100 mL) was treated with pyridine p-toluenesulfonate (2.8 g) overnight.The solution was diluted with water and extracted with dichloromethane.The organic fractions were dried over anhydrous magnesium sulfate,filtered, and the solvent removed to afford the desired crude product(35 g). The crude product was used in the next synthetic step withoutfurther purification.

1,19-Di(tetrahydropyranyloxy)nonadecan-10-ol. A solution of9-tetrahydropyranyloxynonan-1-yl bromide (35 g) in diethyl ether (150mL) was treated with magnesium (3.0 g). A crystal of iodine was added toinitiate the reaction. The solution was refluxed for 4 days, then cooledto room temperature. Ethyl formate (4 mL) was slowly added and thesolution refluxed for 2 hours. The solution was allowed to cool, thenwashed with dilute aqueous sulfuric acid. The organic phase was washedwith water, dried over anhydrous magnesium sulfate, filtered, and thesolvent removed to yield 25 g of crude product. The crude material wasadded to 5% sodium hydroxide in a 1:10 water/methanol solution (150 mL)and heated at 45° C. for one hour. The solution was cooled, diluted withwater and extracted with hexane. The organic fractions were dried overanhydrous magnesium sulfate, filtered and the solvent removed. Theresidue was passed down a silica gel (200 g) column using 0-4%methanol/dichloromethane to afford the desired product (15 g).

1,19-Di(tetrahydropyranyloxy)nonadecan-10-one. A solution of1,19-di(tetrahydropyranyloxy)nonadecan-10-ol (7.2 g) in dichloromethane(40 mL) was treated with pyridinium chlorochromate (4 g) and stirredovernight. Diethyl ether (200 mL) was added and the solution filteredthrough a silica gel bed. The solvent was removed and the residuedissolved in hexane, then filtered through a silica gel bed. The solventwas removed and the residue passed down a silica gel (75 g) column using0-3% methanol/dichloromethane to afford the desired product (3 g).

1,19-Dihydroxynonadecan-10-one. A solution of1,19-di(tetrahydropyranyloxy)nonadecan-10-one (3 g) in methanol (100mL)/water (10 mL) was treated with pyridinium p-toluenesulfonate (1 g)overnight. The solution was filtered to afford the desired product (0.8g). Dilution of the filtrate with water, followed by extraction usingdichloromethane afforded additional desired product (1.0 g).

1,19-Di(2′hexyldecanoyloxy)nonadecan-10-one. A solution of1,19-dihydroxynonadecan-10-one (0.87 g), 2-hexyldecanoic acid (2.48 g),4-dimethylaminopyridine (1.0 g) andN-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (1.68 g) indichloromethane (40 mL) was stirred for three days. The solution wasdiluted with dichloromethane and washed with dilute hydrochloric acid.The organic fraction was dried over anhydrous magnesium sulfate,filtered, and the solvent removed. The residue was passed down a silicagel (50 g) column using dichloromethane to afford the desired product(2.8 g).

1,19-Di(2′hexyldecanoyloxy)nonadecan-10-ol. A solution of1,19-di(2′hexyldecanoyloxy)nonadecan-10-one (1.06 g) in dichloromethane(10 mL) was treated with sodium borohydride (0.15 g). Methanol was addeddropwise until the materials dissolved. The solution was stirred for 30minutes and then partitioned between water and dichloromethane. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel (20 g)column using 0-12% ethyl acetate/hexane gradient to afford the desiredproduct (0.40 g).

10-{[5-(dimethylamino)pentanoyl]oxy}-19-[(2-hexyldecanoyl)oxy]nonadecyl2-hexyldecanoate (Compound 3). A solution of1,19-di(2′hexyldecanoyloxy)nonan-10-ol (0.40 g),5-N,N-dimethylaminopentanoic acid (0.22 g), 4-dimethylaminopyridine(0.18 g) and N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride(0.18 g) in dichloromethane (20 mL) was stirred overnight. The solutionwas diluted with dichloromethane and washed with water. The organicfraction was dried over anhydrous magnesium sulfate, filtered and thesolvent removed. The residue was passed down a silica gel (20 g) columnusing 0-3% methanol/dichloromethane to afford the desired product (0.24g).

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments. These and other changes can be made to the embodiments inlight of the above-detailed description. In general, in the followingclaims, the terms used should not be construed to limit the claims tothe specific embodiments disclosed in the specification and the claims,but should be construed to include all possible embodiments along withthe full scope of equivalents to which such claims are entitled.Accordingly, the claims are not limited by the disclosure.

1. A compound having the following structure (I):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein: one of G¹ or G² is, at each occurrence, —O(C═O)—, —(C═O)O—,—C(═O)—, —O—, —S(O)_(y−), —S—S—, —C(═O)S—, SC(═O)—, —N(R^(a))C(═O)—,—C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—, —OC(═O)N(R^(a))— or—N(R^(a))C(═O)O—, and the other of G¹ or G² is, at each occurrence,—O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(y−), —S—S‘, —C(═O)S—, —SC(═O)—,—N(R^(a))C(═O)—, —C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—,—OC(═O)N(R^(a))— or —N(R^(a))C(═O)O— or a direct bond; L is, at eachoccurrence, ˜O(C═O)—, wherein ˜ represents a covalent bond to X; X isCR^(a); Z is alkyl, cycloalkyl or a monovalent moiety comprising atleast one polar functional group when n is 1; or Z is alkylene,cycloalkylene or a polyvalent moiety comprising at least one polarfunctional group when n is greater than 1; R^(a) is, at each occurrence,independently H, C₁-C₁₂ alkyl, C₁-C₁₂ hydroxylalkyl, C₁-C₁₂ aminoalkyl,C₁-C₁₂ alkylaminylalkyl, C₁-C₁₂ alkoxyalkyl, C₁-C₁₂ alkoxycarbonyl,C₁-C₁₂ alkylcarbonyloxy, C₁-C₁₂ alkylcarbonyloxyalkyl or C₁-C₁₂alkylcarbonyl; R is, at each occurrence, independently either: (a) H orC₁-C₁₂ alkyl; or (b) R together with the carbon atom to which it isbound is taken together with an adjacent R and the carbon atom to whichit is bound to form a carbon-carbon double bond; R¹ and R² have, at eachoccurrence, the following structure, respectively:

a¹ and a² are, at each occurrence, independently an integer from 3 to12; b¹ and b² are, at each occurrence, independently 0 or 1; c¹ and c²are, at each occurrence, independently an integer from 5 to 10; d¹ andd² are, at each occurrence, independently an integer from 5 to 10; y is,at each occurrence, independently an integer from 0 to 2; and n is aninteger from 1 to 6, wherein each alkyl, alkylene, hydroxylalkyl,aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl,alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionallysubstituted with one or more substituent.
 2. The compound of claim 1,wherein G¹ and G² are each independently —O(C═O)— or —(C═O)O—.
 3. Thecompound of claim 1 or 2, wherein X is CH.
 4. The compound of any one ofclaims 1-3, wherein the sum of a¹+b¹+c¹ or the sum of a²+b²+c² is aninteger from 12 to
 26. 5. The compound of any one of claims 1-4, whereina¹ and a² are independently an integer from 3 to
 10. 6. The compound ofany one of claims 1-4, wherein a¹ and a² are independently an integerfrom 4 to
 9. 7. The compound of any one of claims 1-6, wherein b¹ and b²are
 0. 8. The compound of any one of claims 1-6, wherein b¹ and b²are
 1. 9. The compound of any one of claims 1-8, wherein c¹, c², d¹ andd² are independently an integer from 6 to
 8. 10. The compound of any oneof claims 1-9, wherein R is, at each occurrence, independently either:(a) H or methyl; or (b) R together with the carbon atom to which it isbound is taken together with an adjacent R and the carbon atom to whichit is bound to form a carbon-carbon double bond.
 11. The compound of anyone of claims 1-10, wherein each R is H.
 12. The compound of any one ofclaims 1-10, wherein at least one R together with the carbon atom towhich it is bound is taken together with an adjacent R and the carbonatom to which it is bound to form a carbon-carbon double bond.
 13. Thecompound of any one of claims 1-12, wherein R¹ and R² independently haveone of the following structures:


14. The compound of any one of claims 1-13, wherein the compound has oneof the following structures:


15. A compound having the following structure (II):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein: one of G¹ or G² is, at each occurrence, —O(C═O)—, —(C═O)O—,—C(═O)—, —O—, —S(O)_(y−), —S—S—, —C(═O)S—, SC(═O)—, —N(R^(a))C(═O)—,—C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—, —OC(═O)N(R^(a))— or—N(R^(a))C(═O)O—, and the other of G¹ or G² is, at each occurrence,—O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(y−), —S—S—, —C(═O)S—, —SC(═O)—,—N(R^(a))C(═O)—, —C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—,—OC(═O)N(R^(a))— or —N(R^(a))C(═O)O— or a direct bond; L is, at eachoccurrence, ˜O(C═O)—, wherein ˜ represents a covalent bond to X; X isCR^(a); Z is alkyl, cycloalkyl or a monovalent moiety comprising atleast one polar functional group when n is 1; or Z is alkylene,cycloalkylene or a polyvalent moiety comprising at least one polarfunctional group when n is greater than 1; R^(a) is, at each occurrence,independently H, C₁-C₁₂ alkyl, C₁-C₁₂ hydroxylalkyl, C₁-C₁₂ aminoalkyl,C₁-C₁₂ alkylaminylalkyl, C₁-C₁₂ alkoxyalkyl, C₁-C₁₂ alkoxycarbonyl,C₁-C₁₂ alkylcarbonyloxy, C₁-C₁₂ alkylcarbonyloxyalkyl or C₁-C₁₂alkylcarbonyl; R is, at each occurrence, independently either: (a) H orC₁-C₁₂ alkyl; or (b) R together with the carbon atom to which it isbound is taken together with an adjacent R and the carbon atom to whichit is bound to form a carbon-carbon double bond; R¹ and R² have, at eachoccurrence, the following structure, respectively:

R′ is, at each occurrence, independently H or C₁-C₁₂ alkyl; a¹ and a²are, at each occurrence, independently an integer from 3 to 12; b¹ andb² are, at each occurrence, independently 0 or 1; c¹ and c² are, at eachoccurrence, independently an integer from 2 to 12; d¹ and d² are, ateach occurrence, independently an integer from 2 to 12; y is, at eachoccurrence, independently an integer from 0 to 2; and n is an integerfrom 1 to 6, wherein a¹, a², c¹, c², d¹ and d² are selected such thatthe sum of a¹+c¹+d¹ is an integer from 18 to 30, and the sum of a²+c²+d²is an integer from 18 to 30, and wherein each alkyl, alkylene,hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl,alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl andalkylcarbonyl is optionally substituted with one or more substituent.16. The compound of claim 15, wherein G¹ and G² are each independently—O(C═O)— or —(C═O)O—.
 17. The compound of claim 15 or 16, wherein X isCH.
 18. The compound of any one of claims 15-17, wherein the sum ofa¹+b¹+c¹ or the sum of a²+b²+c² is an integer from 12 to
 26. 19. Thecompound of any one of claims 15-18, wherein a¹ and a² are independentlyan integer from 3 to
 10. 20. The compound of any one of claims 15-18,wherein a¹ and a² are independently an integer from 4 to
 9. 21. Thecompound of any one of claims 15-20, wherein b¹ and b² are
 0. 22. Thecompound of any one of claims 15-20, wherein b¹ and b² are
 1. 23. Thecompound of any one of claims 15-22, wherein c¹, c², d¹ and d² areindependently an integer from 6 to
 8. 24. The compound of any one ofclaims 15-23, wherein R is, at each occurrence, independently either:(a) H or methyl; or (b) R together with the carbon atom to which it isbound is taken together with an adjacent R and the carbon atom to whichit is bound to form a carbon-carbon double bond.
 25. The compound of anyone of claims 15-24, wherein each R is H.
 26. The compound of any one ofclaims 15-24, wherein at least one R together with the carbon atom towhich it is bound is taken together with an adjacent R and the carbonatom to which it is bound to form a carbon-carbon double bond.
 27. Thecompound of any one of claims 15-26, wherein each R′ is H.
 28. Thecompound of any one of claims 15-27, wherein the sum of a¹+c¹+d¹ is aninteger from 20 to 25, and the sum of a²+c²+d² is an integer from 20 to25.
 29. The compound of any one of claims 15-28, wherein R¹ and R²independently have one of the following structures:


30. The compound of any one of claims 15-29, wherein the compound hasone of the following structures:


31. The compound of any one of claims 1-30, wherein n is
 1. 32. Thecompound of any one of claims 1-30, wherein n is greater than
 1. 33. Thecompound of any one of claims 1-32, wherein Z is a mono- or polyvalentmoiety comprising at least one polar functional group.
 34. The compoundof claim 33, wherein Z is a monovalent moiety comprising at least onepolar functional group.
 35. The compound of claim 33, wherein Z is apolyvalent moiety comprising at least one polar functional group. 36.The compound of any one of claims 33-35, wherein the polar functionalgroup is a hydroxyl, alkoxy, ester, cyano, amide, amino, alkylaminyl,heterocyclyl or heteroaryl functional group.
 37. The compound of any oneof claims 1-36, wherein Z is hydroxyl, hydroxylalkyl, alkoxyalkyl,amino, aminoalkyl, alkylaminyl, alkylaminylalkyl, heterocyclyl orheterocyclylalkyl.
 38. The compound of any one of claims 1-37, whereinZ, has one of the following structures:


39. The compound of any one of claims 1-38, wherein Z-L has one of thefollowing structures:


40. A compound selected from a compound in Table
 1. 41. A compositioncomprising the compound of any one of claims 1-40 and a therapeuticagent.
 42. The composition of claim 41, further comprising one or moreexcipient selected from neutral lipids, steroids and polymer conjugatedlipids.
 43. The composition of claim 42, wherein the compositioncomprises one or more neutral lipids selected from DSPC, DPPC, DMPC,DOPC, POPC, DOPE and SM.
 44. The composition of claim 43, wherein theneutral lipid is DSPC.
 45. The composition of any one of claims 42-44,wherein the molar ratio of the compound to the neutral lipid ranges fromabout 2:1 to about 8:1.
 46. The composition of any one of claims 42-45,wherein the steroid is cholesterol.
 47. The composition of claim 41,wherein the molar ratio of the compound to cholesterol ranges from 5:1to 1:
 1. 48. The composition of any one of claims 42-47, wherein thepolymer conjugated lipid is pegylated lipid.
 49. The composition ofclaim 48, wherein the molar ratio of the compound to pegylated lipidranges from about 100:1 to about 20:1.
 50. The composition of anyone ofclaim 48 or 49, wherein the pegylated lipid is PEG-DAG, PEG-PE,PEG-S-DAG, PEG-cer or a PEG dialkyoxypropylcarbamate.
 51. Thecomposition of any one of claim 48 or 49, wherein the pegylated lipidhas the following structure (III):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein: R³ and R⁴ are each independently a straight or branched,saturated or unsaturated alkyl chain containing from 10 to 30 carbonatoms, wherein the alkyl chain is optionally interrupted by one or moreester bonds; and w has a mean value ranging from 30 to
 60. 52. Thecomposition of claim 51, wherein R³ and R⁴ are each independentlystraight, saturated alkyl chains containing from 12 to 16 carbon atoms.53. The composition of any one of claim 51 or 52, wherein the average wis about
 45. 54. The composition of any one of claims 41-53, wherein thetherapeutic agent comprises a nucleic acid.
 55. The composition of claim54, wherein the nucleic acid is selected from antisense and messengerRNA.
 56. A method for administering a therapeutic agent to a patient inneed thereof, the method comprising preparing or providing thecomposition of any one of claims 41-55, and administering thecomposition to the patient.